Systems and methods for point of use evacuation of an array

ABSTRACT

Systems, methods, and apparatus are provided for evacuating and for filling an array at the point of use.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Prov. App.Ser. No. 62/510,682 filed on 24 May 2017, the entirety of which isincorporated by reference herein.

BACKGROUND Technical Field

The present disclosure relates generally to apparatus for point of useevacuation and filling of a chamber in a closed system (e.g., an arrayof reaction wells or a reagent blister) and to methods of manufacturingand using the same.

Related Technology

In the United States, Canada, and Western Europe infectious diseaseaccounts for approximately 7% of human mortality, while in developingregions infectious disease accounts for over 40% of human mortality.Infectious diseases lead to a variety of clinical manifestations. Amongcommon overt manifestations are fever, pneumonia, meningitis, diarrhea,and diarrhea containing blood. While the physical manifestations suggestsome pathogens and eliminate others as the etiological agent, a varietyof potential causative agents remain, and clear diagnosis often requiresa variety of assays be performed. Traditional microbiology techniquesfor diagnosing pathogens can take days or weeks, often delaying a propercourse of treatment.

In recent years, the polymerase chain reaction (PCR) has become a methodof choice for rapid diagnosis of infectious agents. PCR can be a rapid,sensitive, and specific tool to diagnose infectious disease. However, achallenge to using PCR as a primary means of diagnosis is the variety ofpossible causative organisms or viruses and the low levels of organismor virus present in some pathological specimens. It is often impracticalto run large panels of PCR assays, one for each possible causativeorganism or viruses, most of which are expected to be negative. Theproblem is exacerbated when pathogen nucleic acid is at lowconcentration and requires a large volume of sample to gather adequatereaction templates. In some cases there is inadequate sample to assayfor all possible etiological agents. A solution is to run “multiplexPCR” wherein the sample is concurrently assayed for multiple targets ina single reaction. While multiplex PCR has proved to be valuable in somesystems, shortcomings exist concerning robustness of high levelmultiplex reactions and difficulties for clear analysis of multipleproducts. To solve these problems, the assay may be subsequently dividedinto multiple secondary PCRs. Nesting secondary reactions within theprimary product increases robustness. Closed systems such as theFilmArray® (BioFire Diagnostics, LLC, Salt Lake City, Utah) reducehandling, thereby diminishing contamination risk.

Technical Problems

A variety of analytical systems and methods incorporate multi-wellarrays as a means of performing analysis on numerous samples. Typically,each well in such as sample vessel is intended to provide a stand-aloneanalysis. Accordingly, the wells and analytical material therein aregenerally designed to be kept separate one from another; without anysubstantial cross-talk therebetween. For this reason, loading each wellof the array presents a challenge, particularly in closed systems. Forexample, in some closed array systems, the wells may be loadedindividually by separate fluid channels that extend to each of the wellsto reduce cross-contamination between the wells. However, filling suchchannels without bubble formation can be challenging, and the presenceof bubbles in the wells can be problematic, as some wells may havediminished sample volume, and the presence of bubbles may lead todetection issues. Additionally, such systems can be costly andcumbersome to manufacture. Moreover, the use of separate fluid channelsmay create well-to-well variability in material and, therefore,analytical results.

In other systems, all or a part of the array may be flooded withmaterial simultaneously and a restrictive opening into each well may beused to reduce contamination between wells. For example, the array canhave a film cover that seals the wells, with a piercing disposed in thefilm over each well. See, for example U.S. Pat. No. 8,895,295, hereinincorporated by reference in its entirety. Fluid material may bedispersed over the surface of the array cover film such that an aliquotof fluid material enters each well through the respective piercings. Thepiercings can be configured to restrict fluid from exiting the well(e.g., absent a force applied thereto).

In various systems, a pressure differential can be used to load thewells with the fluid. For instance, a positive pressure can be used toforce the sample through the fluid pathway, through the piercings, ifapplicable, and into the wells. However, positive pressure applicationsmay damage the structural features associated with the array if pressurein the system builds up. Accordingly, some existing systems that employpositive pressure and separate fluid channels to fill each well use avent or overflow system (e.g. an “out” channel, out of the well), whichallows overfill. However, this method requires a precise measurement ofthe amount of fluid forced into the system and often results inwell-to-well and sample vessel-to-sample vessel variability in filling.In addition, the venting channel may inadvertently introducecontaminants into the array system, which can damage the apparatus andgenerate erroneous results. Moreover, while some of the air disposed inthe fluid pathway is expelled by the incoming fluid, air bubbles canbecome trapped in the pathways, which may affect analysis of the sampleand may enhance cross-talk during analytical methods involvingthermocycling, as the air bubble expands during heating.

As an alternative, negative pressure (or vacuum) can be used to draw thefluid material into each well. Existing systems that employ separatefluid channels to fill each well may evacuate air from the wells andfluid channels by drawing a vacuum through the fluid sample.Accordingly, when the vacuum is released, the fluid material is drawnthrough the fluid channels into each of the wells. However, the level ofvacuum (or pressure differential) is limited to the partial pressure ofthe fluid sample. For example, the partial vapor pressure of water isabout 25-32 millibar at room-temperature (i.e., 20-25° C.) and at 1atmosphere of pressure, and the partial vapor pressure of water rises asa vacuum is drawn. Accordingly, only a limited amount of air can beremoved from each well in the presence of water or other fluid.

In existing array flooding systems, the vacuum may be applied or drawnat the time of manufacture. For example, an array can be sealed in anevacuation chamber such that the array is assembled under vacuum (ornegative pressure) conditions. The array system is then packaged,stored, and transported under conditions that maintain the vacuumapplied during manufacture. However, such a system requires materialsand/or packaging that can maintain an appropriate level of vacuum untiluse, which can add considerable expense to the product.

Accordingly, there exists a need in the art for products, methods, andsystems for in situ evacuation and filling of chambers in a closedsystem (e.g., an array of reaction wells or a reagent blister), a needfor instruments configured for in situ evacuation of the chambers in aclosed system, a need for methods of use for reaction chambers thatemploy in situ evacuation, and a need for methods for manufacturingreaction containers configured for in situ evacuation.

BRIEF SUMMARY Solutions to the Problems

Embodiments of the present disclosure solve one or more of the foregoingor other problems in the art with novel systems and methods forpoint-of-use evacuation and, preferably, subsequent filling of one ormore types of chambers, especially in a closed environment. For example,some embodiments include an array assembly that can be evacuated ondemand, at the point-of-use, and/or in real-time (i.e., duringperformance of steps of an analytical method). Accordingly, in preferredembodiments, the vacuum need only be held for a few seconds to a fewminutes (i.e., long enough to draw the vacuum, reseal the system, and,preferably, open a fill seal to fill the evacuated chamber (e.g., anarray of wells) with a liquid). Thus, a near fully drawn vacuum can beused to draw fluid into the array assembly upon opening the fill seal.

Because, in one illustrative example, the vacuum is not drawn through aliquid sample, the vacuum is not limited to the vapor pressure of theliquid (e.g., water). The negative pressure level of the strong vacuummay thoroughly evacuate air from the array assembly so that residual airis minimized in the array assembly. Illustratively, the strong vacuumalso effectively draws a full, consistent amount of fluid into each ofthe wells upon releasing the vacuum. Because air in the array assemblyhas been thoroughly evacuated, air bubbles in the drawn fluid may bereduced, as compared to existing systems, minimized, or, preferably,eliminated. Moreover, the array assembly can be configured (e.g., sizedand structured) so that an evacuation chamber is not required to applythe vacuum. Instead, in illustrative embodiments, a piston pump or othervacuum device can be used to apply closer to a true vacuum in the arrayassembly.

In some embodiments, the array assembly can have a plurality of wellsconfigured in an array, a vacuum channel in fluid communication with theplurality of wells and with a vacuum port, and an array fill channel influid communication with the plurality of wells and with a fluidreservoir. The fluid channel and the vacuum channel may be co-alignedand may be co-extensive, and, in some embodiment, may be the same. Anopenable fill seal can be disposed in the fill channel or a fluidchannel disposed between the fill channel and the fluid reservoir. Insome embodiments, the fill seal can be or comprise an openable seal(e.g., a frangible seal or a peelable seal), such as a seal formed bythe collapsing the fill channel or fluid channel. A vacuum device can becoupled with the vacuum port and a vacuum can be applied to the arrayassembly (i.e., vacuum channel, plurality of wells, and fill channel.

A vacuum seal can be preferably placed over the vacuum channel (e.g.,between the vacuum port and the plurality of wells) to maintain thevacuum in the wells. The openable fill seal, which may be preferablyplaced between the array and another blister (e.g., a fluid reservoir),can prevent evacuation of the fluid reservoir while the array assemblyis under vacuum. When the fill seal is opened, fluid in the reservoircan be drawn through the fluid channel and array fill channel, into theplurality of wells. In the plurality of wells, the fluid can mix withone or more reagents.

In some embodiments, the well seal can be formed between at least someof the plurality of wells. For instance, segments of the array fillchannel can extend through a row of wells in the array. Accordingly, thefill channel segment can communicate with wells in the row, but notdirectly with wells in an adjacent or different row. By forming a wellseal, such as a heat weld between the card layer and the second filmlayer, crossways of the row of wells and the fill channel segments, eachwell can be sealed off or isolated from all other wells. In this way,cross-talk between wells of the array can be minimized or eliminated.

In some embodiments, an openable fill seal can also be formed in thefluid channel in fluid communication with the fluid reservoir and thefill channel. Because the array assembly need not be assembled undervacuum, such as in a vacuum chamber, the array assembly need not bepackaged, stored, and shipped in a vacuum-sealed container. Thisadvantage of the present disclosure can produce significant labor andmaterial cost savings, making analytical techniques performed with thearray assembly more affordable for consumers. Moreover, embodiments ofthe present disclosure can have a smaller material footprint, as robust,vacuum-sealable packaging containers need not be used.

In some embodiments, the array assembly can include a card layer havingthe plurality of wells formed therein, a first film layer bonded to afirst side of the card layer, and/or a second film layer bonded to asecond side of the card layer. The first film layer can seal a first endof each of the plurality of wells. The second film layer can at leastpartially seal a second end of the plurality of wells. The first filmlayer and second film layer can form a pouch in which the card layer isdisposed. For instance, the card layer can be laminated by adhesive orthermoforming between the first film layer and second film layer.

In certain embodiments, the card layer, first film layer, and/or secondfilm layer can be configured to and/or cooperate to form the vacuumchannel and/or the array fill channel. For instance, in at least oneembodiment, the vacuum channel and/or the fill channel can be formed inthe card layer or can, for example, be impressed into a surface portionthereof. In some embodiments, the vacuum channel and/or the fill channelcan be formed in the second film layer or surface portion thereof. Thesecond film layer can be bonded to a third film layer such that thevacuum channel and/or the fill channel are formed between the secondfilm layer and the third film layer. The third film layer can have aplurality of piercings. The piercings can be aligned and/or in fluidcommunication with the plurality of wells, the vacuum channel, and/orthe fill channel.

The array assembly can be manufactured by bonding the first film layerto the first side of a card layer and bonding the second film layer to asecond side of the card layer so as to form the vacuum channel and thearray fill channel. In some embodiments, the first film layer and thesecond film layer can be bonded together forming a card layer pouch.Bonding the film layers together can form the fluid channel, liquidreservoir, and/or other component(s) of a sample vessel. The card layer(e.g., with recessed conduit formed therein) can optionally be preparedwith reagents in the plurality of wells and inserted into the pouchthrough an opening formed by separation of the first and second filmlayers. The first and second film layers can be bonded to opposing sidesof the card, and the pouch can be sealed with the card layer therein,such as by heat welding the opening in the pouch.

Described herein are:

1. An array assembly, comprising:

a first layer and at least a second layer;

a third layer having a plurality of wells formed therein, the thirdlayer being disposed between the first and second layers;

a vacuum channel in fluid communication with the plurality of wells andwith a vacuum port; and

an array fill channel in fluid communication with the plurality of wellsand with a fluid source.

2. The array assembly of clause 1, wherein the first and second layersare film layers.

3. The array assembly of clauses 1 or 2, wherein the third layercomprises a card layer having the plurality of wells formed therein.

4. The array assembly of clause 3, wherein the first layer is bonded toa first side of the card layer and seals a first end of each of theplurality of wells, and the second layer is bonded to a second side ofthe card layer and seals a second end of each of the plurality of wells.

5. The array assembly of clauses 1-4, wherein the vacuum port comprisesan opening in one or more of the first layer or the second layer.

6. The array assembly of clauses 3-5, further comprising a recessedconduit in the card layer, wherein at least a portion of one or more ofthe vacuum channel and the array fill channel is formed between thesecond film layer and the recessed conduit in the card layer, andwherein the recessed conduit in the card layer has a manifoldconfiguration that fluidically connects the plurality of wells to thevacuum port by at least two paths and fluidically connects each well tothe fluid source by at least two paths.

7. The array assembly of clauses 1-6, wherein at least a portion of thevacuum channel is disposed in and/or co-localized with at least aportion of the array fill channel.

8. The array assembly of clauses 1-7, further comprising an openableseal in the array fill channel between the plurality of wells and thefluid source.

9. The array assembly of clauses 1-8, wherein the plurality of wells aredisposed in a respective plurality of rows, the array fill channelcomprises an access channel in fluid communication with the fluid sourceand with a manifold channel assembly, the manifold channel assemblycomprising:

a plurality of branch channels extending along the respective pluralityof rows, the plurality of branch channels respectively being in fluidcommunication with the plurality of wells; and

a first main channel extending along a first end of the plurality ofrows, the plurality of branch channels extending from the first mainchannel, the array fill channel in communication with the first mainchannel.

10. The array assembly of clauses 1-9, wherein the manifold channelassembly further comprises a second main channel in fluid communicationwith the plurality of branch channels, the second main channel extendingalong a second end of the plurality of rows, the vacuum port incommunication with the second main channel, wherein each well is influid communication with the vacuum port and the fluid source by atleast two different paths.

11. The array assembly of clauses 1-10, wherein the manifold channelassembly further comprises a plurality of connection channels extendingrespectively from each of the plurality of branch channels to theplurality of wells, the plurality of connection channels respectivelybeing in fluid communication with the plurality of wells.

12. A method of manufacturing an array assembly, the method comprising:

providing a card layer having a plurality of wells disposed therein;

disposing the card layer between a first film layer and at least asecond film layer;

bonding the first film layer to a first side of a card layer; and

bonding the second film layer to a second side of the card layer,

wherein the card layer and at least one of the first or second filmlayers form:

(i) a vacuum channel extending between and/or in fluid communicationwith the plurality of wells and a vacuum port; and

(ii) an array fill channel extending between and/or in fluidcommunication with the plurality of wells and a fluid source.

13. The method of clause 12, wherein the recessed conduit in the cardlayer has a manifold configuration.

14. A method of using an array assembly on a sample, comprising

(a) providing a sample container comprising a reaction zone in fluidcommunication with the array assembly, the array assembly comprising

a plurality of wells configured in an array,

an access opening between the reaction zone and array,

a vacuum port,

a plurality of channels such that each well in the array is fluidlyconnected to the access opening and the vacuum port;

(b) performing an analytical method on the sample in the reaction zoneto produce a reaction mixture;

(c) opening the vacuum port and drawing a vacuum on the array assemblysuch that air is evacuated from the plurality of wells and the pluralityof channels;

(d) sealing vacuum port such that the plurality of wells are maintainedunder the vacuum, thereby forming an evacuated array; and

(e) opening the access opening such that the reaction mixture is drawninto the plurality of wells via the array fill channel.

15. The method of clause 14, further comprising mixing the reactionmixture with one or more reagents disposed in each of the plurality ofwells to form a second mixture, and performing a second analyticalmethod on the second reaction mixture.

16. The method of clause 14 or 15, wherein the second mixture is a PCRmixture.

17. The method of clauses 14-16, further comprising thermocycling thesecond reaction mixture.

18. The method of clauses 14-17, further comprising detecting anamplification product in at least one of the plurality of wells.

19. The method of clauses 14-18, wherein the sample comprises amicroorganism and one or more reagents comprises an antibiotic.

20. The method of clause 19, wherein the antibiotic is present in afirst of the plurality of wells at a first concentration and is presentin a second of the plurality of wells at a second concentration, whereinthe second concentration is lower than the first concentration.

21. The method of clauses 14-20, wherein step (c) provides a vacuum inthe plurality of wells of between about 2 millibar and about 150millibar.

22. The method of clauses 14-21, wherein steps (c) and (d) are performedbefore step (b) is completed.

23. A method for performing a multi-step biological reaction in a sealedsample container, comprising:

(a) providing a sealed container comprising a first reaction zone, asecond reaction zone, and dried components in the second reaction zone,

(b) performing a first reaction in the first reaction zone, to generatea reaction mixture,

(c) hydrating the dried components in the second reaction zone with ahydration fluid to generate hydrated components, wherein step (c) isperformed before or during step (b),

(d) adding a portion of the reaction mixture to the hydrated components,and

(e) performing a second reaction in the second reaction zone.

24. The method of clause 23, wherein the second reaction zone is anarray of wells, wherein each well is provided with dried components, andwherein at least a plurality of wells has components that are differentfrom other wells in the plurality of wells.

25. The method of clauses 23 or 24, wherein each individual well in thearray of wells is provided with a barrier layer that retards entry ofthe hydration fluid into the wells.

26. The method of clauses 23-25, wherein the hydrating step comprisesfilling the plurality of wells with the hydration fluid, followed byremoving from the array of wells of any excess hydration fluid.

27. The method of clauses 23-26, wherein the adding step comprisesmoving the reaction mixture to the array, allowing a portion of thereaction mixture to cross the barrier layer and enter each well, andremoving from the array any excess reaction mixture.

28. The method of clauses 23-26, wherein the adding step comprisesmoving the reaction mixture to the array, and sealing a portion of thereaction mixture adjacent each well outside the barrier layer, so thatthe reaction mixture and hydrated components may mix through the barrierlayer.

29. The method of clauses 23-28, wherein the barrier layer is a piercedlayer having one or more openings per well, wherein the openings aresized such that absent some force, fluid does not readily flow throughthe openings.

30. The method of clauses 23-29, wherein the first reaction isfirst-stage PCR and the second reaction is second-PCR.

31. The method of clauses 23-30, wherein the second reaction zone ismaintained at a cool temperature prior to step (d).

32. The method of clauses 23-31, further comprising evacuating at leastthe second reaction zone with a partial vacuum prior to step (c).

33. An array assembly, comprising:

a card having a plurality of wells arranged in an array;

an access opening in a first side of the card;

a channel system in fluid communication with the access opening and withthe plurality of wells; and

a vacuum port in fluid communication with the channel system.

34. The array assembly of clause 33, wherein the array comprises aplurality of rows, each of the plurality of rows comprising one or morewells, the channel system comprising a manifold channel assemblycomprising:

a plurality of branch channels extending along the plurality of rows,the plurality of branch channels respectively being in fluidcommunication with the plurality of wells; and

a first main channel in fluid communication with the access openingextending along a first end of the plurality of rows, the plurality ofbranch channels extending from the first main channel,

a second main channel in fluid communication with the plurality ofbranch channels, the second main channel extending along a second end ofthe plurality of rows, the vacuum port in communication with the secondmain channel,

wherein each well is in fluid communication with the vacuum port and theaccess opening by at least two different paths.

35. The array assembly of one of clauses 33 or 34, further comprising afirst film layer and a second film layer, wherein first and second filmlayers form a pouch, the card layer being at least partially sealedinside the pouch.

36. A reaction container, comprising:

a plurality of fluidly connected reaction chambers,

an array comprising

an access opening in fluid communication with at least one of thefluidly connected reaction chambers;

a plurality of reaction wells;

a vacuum port;

a channel system in fluid communication with the access opening, withthe plurality of wells and with the vacuum port, the channel systemproviding paths such that each reaction well in the array is fluidlyconnected to the vacuum port by at least two paths and each reactionwell is also connected to the fluid source by at least two paths.

37. The array assembly of clause 36, wherein the vacuum port isreversibly sealable and the access opening is reversibly sealable.

38. An array assembly, comprising:

a plurality of wells arranged in an array;

a vacuum channel in fluid communication with the plurality of wells andwith a vacuum port; and

an array fill channel in fluid communication with the plurality of wellsand with a fluid source.

39. The array assembly of clause 38, further comprising

a card layer having the plurality of wells formed therein,

a first film layer bonded to a first side of the card layer, wherein thefirst film layer seals a first end of each of the plurality of wells,

a second film layer bonded to a second side of the card layer, whereinthe vacuum port comprises an opening in one or more of the first filmlayer and the second film layer.

40. The array assembly of clause 38 or 39, wherein at least a portion ofone or more of the vacuum channel and the array fill channel is formed(i) in the card layer, (ii) in the second film layer, or (iii) betweenthe card layer and the second film layer.

41. The array assembly of clauses 38-40, wherein at least a portion ofone or more of the vacuum channel and the array fill channel is formedbetween the first film layer and the second film layer.

42. The array assembly of clauses 38-41, wherein a portion of the arrayfill channel is formed between the first film layer and the second filmlayer and is disposed about at least a portion of a perimeter of thecard layer.

43. The array assembly of clauses 38-42, further comprising a recessedconduit in the card layer, wherein the recessed conduit in the cardlayer has a manifold configuration wherein each well in the array isfluidly connected to the vacuum port by at least two paths and each wellis also connected to the fluid source by at least two paths.

44. The array assembly of clauses 38-43, wherein the second film layerhas a recessed conduit formed therein, further comprising a third filmlayer disposed between the second film layer and the card layer, whereinthe third film layer has a plurality of piercings extendingtherethrough, and wherein the plurality of piercings are in fluidcommunication with the recessed conduit in the second film layer.

45. The array assembly of clauses 38-44, wherein at least a portion ofthe array fill channel is disposed between the recessed conduit in thesecond film layer and the bond between the third film layer and thesecond film layer between two or more portions of the recessed conduitin the second film layer.

46. The array assembly of clauses 38-45, wherein at least a portion ofthe vacuum channel is provided as a portion of the array fill channeland is configured to maintain the array fill channel in an openposition.

47. The array assembly of clauses 38-46, further comprising an openableseal in the array fill channel between the plurality of wells and thefluid source.

48. The array assembly of clauses 38-47, further comprising one or morereagents disposed in each of the plurality of wells.

49. The array assembly of clauses 38-48, wherein the plurality of wellsare disposed in a respective plurality of rows, the array fill channelcomprising an access channel in fluid communication with the fluidsource and with a manifold channel assembly, the manifold channelassembly comprising:

a plurality of branch channels extending along the respective pluralityof row, the plurality of branch channels respectively being in fluidcommunication with the plurality of wells; and

a first main channel extending along a first end of the plurality ofrows, the plurality of branch channels extending from the first mainchannel, the array fill channel extending from the first main channel.

50. The array assembly of clauses 38-49, wherein the manifold channelassembly further comprises a second main channel in fluid communicationwith the plurality of branch channels, the second main channel extendingalong a second end of the plurality of rows, the vacuum port incommunication with the second main channel, wherein each well is influid communication with the vacuum port and the fluid source by atleast two different paths.

51. The array assembly of clauses 38-50, wherein the manifold channelassembly further comprises a plurality of connection channels extendingrespectively from each of the plurality of branch channels to theplurality of wells, the plurality of connection channels respectivelybeing in fluid communication with the plurality of wells.

52. A method of drawing a vacuum on a reaction container in situ whileperforming an analytical method, the method comprising:

providing a reaction container that includes a sample introduction zone,at least a first reaction zone in fluid communication with the sampleintroduction zone, and a second reaction zone in fluid communicationwith the first reaction zone, wherein the second reaction zone comprisesa plurality of wells, a vacuum channel in fluid communication with theplurality of wells and a vacuum port, an array fill channel extendingbetween and/or in fluid communication with the plurality of wells andthe first reaction zone, and an openable seal disposed between the firstreaction zone and the second reaction zone;

performing at least one step of the analytical method with the reactioncontainer;

drawing a partial vacuum on the plurality of wells such that theplurality of wells, the vacuum channel, and at least a portion of thearray fill channel are under reduced pressure relative to atmosphericpressure;

sealing a portion of the vacuum channel such that the plurality of wellsand at least the portion of the array fill channel are maintained underthe vacuum, thereby forming an evacuated array; and

applying a fluid to the evacuated array by opening the openable sealdisposed between the first reaction zone and the second reaction zonesuch that the fluid is drawn into the plurality of wells via the arrayfill channel.

53. The method of clause 52, wherein the at least one step of theanalytical method includes removing the reaction container from anambient pressure package, introducing a sample into the sampleintroduction zone, inserting the reaction container into an instrumentconfigured for performing the analytical method and configured fordrawing a partial vacuum to form the evacuated array, performing one ormore reactions in the first reaction zone, or preparing for applying thefluid to the evacuated array.

54. The method of clauses 52 or 53, wherein performing one or morereactions in the first reaction zone comprises one or more of performinga sample lysis to generate a lysate, isolating lysis particles from thelysate, mixing silica magnetic beads with the lysate, moving a residuallysate after nucleic acid capture with the silica magnetic beads to awaste chamber, performing at least one wash of the magnetic beads andmoving the wash liquid to the waste chamber, eluting nucleic acids fromthe silica magnetic beads, performing a first singleplex or multiplexPCR reaction, or diluting a product of the first PCR reaction inpreparation for performing a second PCR reaction in the plurality ofwells of the second reaction zone.

55. The method of clauses 52-54, wherein the reaction container furtherincludes one or more reagent blisters fluidically connected to one ormore of the sample introduction zone, the first reaction zone, or thesecond reaction zone.

56. The method of clauses 52-55, wherein at least one of the one or morereagent blisters includes a dried reagent, and the method furthercomprising drawing a partial vacuum on the one or more reagent blistersincluding the dried reagent.

57. The method of clauses 52-56, further comprising mixing a fluid withthe dried reagent to form a first mixture.

58. The method of clauses 52-57, wherein the one or more reagentblisters comprise reagents for sample preparation, nucleic acidrecovery, a first-stage PCR, and a second-stage PCR.

59. A system, comprising:

a reaction container that includes a sample introduction zone, at leasta first reaction zone in fluid communication with the sampleintroduction zone, and a second reaction zone in fluid communicationwith the first reaction zone, wherein the second reaction zone comprisesa plurality of wells, a vacuum channel extending between and/or in fluidcommunication with the plurality of wells and a vacuum port, an arrayfill channel extending between and/or in fluid communication with theplurality of wells and the first reaction zone, and an openable sealdisposed between the first reaction zone and the second reaction zone;

an instrument configured to perform an analytical method using thereaction container, wherein the instrument includes a vacuum system todraw a partial vacuum in one or more portions of the reaction containerwhile performing one or more steps of the analytical method.

60. The system of clause 59, wherein the one or more portions of thereaction container configured to have a partial vacuum drawn thereon aresubstantially dry such that the partial vacuum is not being drawnagainst a partial pressure of water.

61. The system of clauses 59 or 60, wherein the partial vacuum in theone or more portions of the reaction container configured to have apartial vacuum drawn thereon is in a range between about 2 millibar andabout 150 millibar.

62. The system of clauses 59-61, wherein the one or more portions of thereaction container configured to have a partial vacuum drawn thereoninclude the plurality of wells, the vacuum channel, and at least aportion of the array fill channel.

63. The system of clauses 59-62, wherein the instrument includes a sealdevice apply a seal to preserve the partial vacuum drawn one or moreportions of the reaction container.

64. The system of clauses 59-63, wherein the reaction container furtherincludes one or more reagent blisters fluidically connected to one ormore of the sample introduction zone, the first reaction zone, or thesecond reaction zone.

65. The system of clauses 59-64, wherein at least one of the one or morereagent blisters includes a dried reagent, and wherein the one or moreportions of the reaction container configured to have a partial vacuumdrawn thereon include the one or more reagent blisters that include thedried reagent.

66. The system of clauses 59-65, wherein the instrument is a PCRinstrument that includes at least one heater positioned and arranged forthermocycling at least one portion of the reaction container.

67. The system of clauses 59-66, wherein the instrument includes atleast one heater positioned and arranged for controlling the temperaturein at least one portion of the reaction container for performing anisothermal reaction.

68. The system of clauses 59-67, wherein the instrument includes atleast one heater positioned and arranged for controlling the temperaturein at least one portion of the reaction container, one or more actuatorsfor moving fluid in the reaction container, and one or more seals forcontrolling the movement of fluids within one or more portions of thereaction container.

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used as an aid in determining the scope of the claimed subjectmatter.

Additional features and advantages will be set forth in the descriptionthat follows, and in part will be obvious from the description, or maybe learned by the practice of the invention. The features and advantagesmay be realized and obtained by means of the instruments andcombinations particularly pointed out in the appended claims. These andother features will become more fully apparent from the followingdescription and appended claims, or may be learned by the practice ofthe invention as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flexible pouch useful for self-contained PCR.

FIG. 2 is an exploded perspective view of an instrument for use with thepouch of FIG. 1, including the pouch of FIG. 1.

FIG. 3 shows a partial cross-sectional view of the instrument of FIG.3A, including the bladder components of FIG. 2, with the pouch of FIG.1.

FIG. 4 shows a motor used in one illustrative embodiment of theinstrument of FIG. 2.

FIG. 5A shows another embodiment of a flexible pouch.

FIG. 5B illustrates a cross-sectional view of a portion of the pouch ofFIG. 5A along the line B-B.

FIG. 5C illustrates a cross-sectional view of a portion of the pouch ofFIG. 5A along the line C-C.

FIG. 6A shows an illustrative second-stage array.

FIG. 6B is a cross-sectional view illustrated along the line B-B of FIG.6A showing one embodiment of a well filling system.

FIG. 6C is an alternative embodiment showing a different embodiment of awell filling system.

FIG. 7A illustrates a second-stage array showing one embodiment of apoint-of-use array evacuation system.

FIG. 7B is a cross-sectional view of a portion of FIG. 7A along the lineB-B.

FIG. 8A is a perspective view of one illustrative embodiment of an arrayassembly comprising a card.

FIG. 8B is a detailed view of a portion of the array assembly of FIG.8A.

FIG. 8C is a top plan view of another illustrative embodiment of anarray assembly comprising a card layer in a pouch.

FIG. 8D is a top plan view of a pouch.

FIG. 9A is an exploded perspective view of an embodiment of asecond-stage high density array having a vacuum channel.

FIG. 9B is a bottom view of the second-stage high density array of FIG.9A, shown during construction of the second-stage high density array.

FIG. 9C is a cross-sectional view of a portion of FIG. 9A along the lineC-C.

FIGS. 10A-10E illustrate a method for rehydrating the contents andinitiating a reaction in an illustrative well of a second-stage array.

FIG. 10F illustrates an alternative method of initiating a reaction inthe method illustrated in FIGS. 10A-10E.

FIG. 11A schematically illustrates an instrument configured for drawinga vacuum on a portion of a reaction container during or after performingone or more steps of an analytical method.

FIG. 11B illustrates the vacuum system and a portion of an array of thereaction container of FIG. 11A configured for in situ drawing of avacuum.

FIG. 11C illustrates the vacuum system and a reagent blister of thereaction container of FIG. 11A configured for in situ drawing of avacuum.

FIGS. 12A-12D show various array well and channel configurations.

DETAILED DESCRIPTION

Example embodiments are described below with reference to theaccompanying drawings. Many different forms and embodiments are possiblewithout deviating from the spirit and teachings of this disclosure andso the disclosure should not be construed as limited to the exampleembodiments set forth herein. Rather, these example embodiments areprovided so that this disclosure will be thorough and complete, and willconvey the scope of the disclosure to those skilled in the art. In thedrawings, the sizes and relative sizes of layers and regions may beexaggerated for clarity. Like reference numbers refer to like elementsthroughout the description.

Unless defined otherwise, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which the present disclosure pertains.It will be further understood that terms, such as those defined incommonly used dictionaries, should be interpreted as having a meaningthat is consistent with their meaning in the context of the presentapplication and relevant art and should not be interpreted in anidealized or overly formal sense unless expressly so defined herein. Theterminology used in the description of the invention herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting of the invention. While a number of methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present disclosure, only certain exemplary materials andmethods are described herein.

All publications, patent applications, patents or other referencesmentioned herein are incorporated by reference for in their entirety. Incase of a conflict in terminology, the present specification iscontrolling.

Various aspects of the present disclosure, including devices, systems,methods, etc., may be illustrated with reference to one or moreexemplary implementations. As used herein, the terms “exemplary” and“illustrative” mean “serving as an example, instance, or illustration,”and should not necessarily be construed as preferred or advantageousover other implementations disclosed herein. In addition, reference toan “implementation” or “embodiment” of the present disclosure orinvention includes a specific reference to one or more embodimentsthereof, and vice versa, and is intended to provide illustrativeexamples without limiting the scope of the invention, which is indicatedby the appended claims rather than by the following description.

It will be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a tile” includes one, two, or more tiles. Similarly,reference to a plurality of referents should be interpreted ascomprising a single referent and/or a plurality of referents unless thecontent and/or context clearly dictate otherwise. Thus, reference to“tiles” does not necessarily require a plurality of such tiles. Instead,it will be appreciated that independent of conjugation; one or moretiles are contemplated herein.

As used throughout this application the words “can” and “may” are usedin a permissive sense (i.e., meaning having the potential to), ratherthan the mandatory sense (i.e., meaning must). Additionally, the terms“including,” “having,” “involving,” “containing,” “characterized by,”variants thereof (e.g., “includes,” “has,” “involves,” “contains,”etc.), and similar terms as used herein, including the claims, shall beinclusive and/or open-ended, shall have the same meaning as the word“comprising” and variants thereof (e.g., “comprise” and “comprises”),and do not exclude additional, un-recited elements or method steps,illustratively.

As used herein, directional and/or arbitrary terms, such as “top,”“bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “inner,”“outer,” “internal,” “external,” “interior,” “exterior,” “proximal,”“distal,” “forward,” “reverse,” and the like can be used solely toindicate relative directions and/or orientations and may not beotherwise intended to limit the scope of the disclosure, including thespecification, invention, and/or claims.

It will be understood that when an element is referred to as being“coupled,” “connected,” or “responsive” to, or “on,” another element, itcan be directly coupled, connected, or responsive to, or on, the otherelement, or intervening elements may also be present. In contrast, whenan element is referred to as being “directly coupled,” “directlyconnected,” or “directly responsive” to, or “directly on,” anotherelement, there are no intervening elements present.

Example embodiments of the present inventive concepts are describedherein with reference to cross-sectional illustrations that areschematic illustrations of idealized embodiments (and intermediatestructures) of example embodiments. As such, variations from the shapesof the illustrations as a result, for example, of manufacturingtechniques and/or tolerances, are to be expected. Thus, exampleembodiments of the present inventive concepts should not be construed aslimited to the particular shapes of regions illustrated herein but areto include deviations in shapes that result, for example, frommanufacturing. Accordingly, the regions illustrated in the figures areschematic in nature and their shapes are not intended to illustrate theactual shape of a region of a device and are not intended to limit thescope of example embodiments.

It will be understood that although the terms “first,” “second,” etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. Thus, a “first” element could be termed a“second” element without departing from the teachings of the presentembodiments.

It is also understood that various implementations described herein canbe utilized in combination with any other implementation described ordisclosed, without departing from the scope of the present disclosure.Therefore, products, members, elements, devices, apparatuses, systems,methods, processes, compositions, and/or kits according to certainimplementations of the present disclosure can include, incorporate, orotherwise comprise properties, features, components, members, elements,steps, and/or the like described in other implementations (includingsystems, methods, apparatus, and/or the like) disclosed herein withoutdeparting from the scope of the present disclosure. Some embodiments, oraspects thereof, may be described as alternatives. It will beappreciated, however, that such alternatives may not always be mutuallyexclusive. Accordingly, the terms “alternative,” “alternatively,” andthe like can be replaced with “additional,” “additionally,” and thelike. Thus, reference to a specific feature in relation to oneimplementation should not be construed as being limited to applicationsonly within that implementation.

The headings used herein are for organizational purposes only and arenot meant to be used to limit the scope of the description or theclaims. To facilitate understanding, like reference numerals have beenused, where possible, to designate like elements common to the figures.Furthermore, where possible, like numbering of elements have been usedin various figures. Furthermore, alternative configurations of aparticular element may each include separate letters appended to theelement number.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 5%. When such a range is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

By “sample” is meant an animal; a tissue or organ from an animal; a cell(either within a subject, taken directly from a subject, or a cellmaintained in culture or from a cultured cell line); a cell lysate (orlysate fraction) or cell extract; a solution containing one or moremolecules derived from a cell, cellular material, or viral material(e.g. a polypeptide or nucleic acid); or a solution containing anon-naturally occurring nucleic acid, which is assayed as describedherein. A sample may also be any body fluid or excretion (for example,but not limited to, blood, urine, stool, saliva, tears, bile, orcerebrospinal fluid) that may or may not contain host or pathogen cells,cell components, or nucleic acids. Samples may also includeenvironmental samples such as, but not limited to, soil, water (freshwater, waste water, etc.), air monitoring system samples (e.g., materialcaptured in an air filter medium), surface swabs, and vectors (e.g.,mosquitos, ticks, fleas, etc.).

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages). In particular, nucleic acids can include, withoutlimitation, DNA, RNA, mRNA, rRNA, cDNA, gDNA, ssDNA, dsDNA, or anycombination thereof.

By “probe,” “primer,” or “oligonucleotide” is meant a single-strandednucleic acid molecule of defined sequence that can base-pair to a secondnucleic acid molecule that contains a complementary sequence (the“target”). The stability of the resulting hybrid depends upon thelength, GC content, and the extent of the base-pairing that occurs. Theextent of base-pairing is affected by parameters such as the degree ofcomplementarity between the probe and target molecules and the degree ofstringency of the hybridization conditions. The degree of hybridizationstringency is affected by parameters such as temperature, saltconcentration, and the concentration of organic molecules such asformamide, and is determined by methods known to one skilled in the art.Probes, primers, and oligonucleotides may be detectably-labeled, eitherradioactively, fluorescently, or non-radioactively, by methodswell-known to those skilled in the art. dsDNA binding dyes may be usedto detect dsDNA. It is understood that a “primer” is specificallyconfigured to be extended by a polymerase, whereas a “probe” or“oligonucleotide” may or may not be so configured.

By “dsDNA binding dyes” is meant dyes that fluoresce differentially whenbound to double-stranded DNA than when bound to single-stranded DNA orfree in solution, usually by fluorescing more strongly. While referenceis made to dsDNA binding dyes, it is understood that any suitable dyemay be used herein, with some non-limiting illustrative dyes describedin U.S. Pat. No. 7,387,887, herein incorporated by reference. Othersignal producing substances may be used for detecting nucleic acidamplification and melting, illustratively enzymes, antibodies, etc., asare known in the art.

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a sample nucleic acid) under high stringency conditions, anddoes not substantially base pair with other nucleic acids.

By “high stringency conditions” is meant typically to occur at about amelting temperature (Tm) minus 5° C. (i.e. 5° below the Tm of theprobe). Functionally, high stringency conditions are used to identifynucleic acid sequences having at least 80% sequence identity.

While PCR is the amplification method used in the examples herein, it isunderstood that any amplification method that uses a primer may besuitable. Such suitable procedures include polymerase chain reaction(PCR); strand displacement amplification (SDA); nucleic acidsequence-based amplification (NASBA); cascade rolling circleamplification (CRCA), loop-mediated isothermal amplification of DNA(LAMP); isothermal and chimeric primer-initiated amplification ofnucleic acids (ICAN); target based-helicase dependent amplification(HDA); transcription-mediated amplification (TMA), and the like.Therefore, when the term PCR is used, it should be understood to includeother alternative amplification methods. For amplification methodswithout discrete cycles, reaction time may be used where measurementsare made in cycles, doubling time, or crossing point (Cp), andadditional reaction time may be added where additional PCR cycles areadded in the embodiments described herein. It is understood thatprotocols may need to be adjusted accordingly.

As used herein, the term “crossing point” (Cp) (or, alternatively, cyclethreshold (Ct), quantification cycle (Cq), or a synonymous term used inthe art) refers to the number of cycles of PCR required to obtain afluorescence signal above some threshold value for a given PCR product(e.g., target or internal standard(s)), as determined experimentally.The cycle where each reaction rises above the threshold is dependent onthe amount of target (i.e., reaction template) present at the beginningof the PCR reaction. The threshold value may typically be set at thepoint where the product's fluorescence signal is detectable abovebackground fluorescence; however, other threshold values may beemployed. As an alternative to setting a somewhat arbitrary thresholdvalue, Cp may be determined by calculating the point for a reaction atwhich a first, second, or nth order derivative has its maximum value,which determines the cycle at which the curvature of the amplificationcurve is maximal. An illustrative derivative method was taught in U.S.Pat. No. 6,303,305, herein incorporated by reference in its entirety.Nevertheless, it usually does not matter much where or how the thresholdis set, so long as the same threshold is used for all reactions that arebeing compared. Other points may be used as well, as are known in theart, and any such point may be substituted for Cp, Ct, or Cq in any ofthe methods discussed herein.

While various examples herein reference human targets and humanpathogens, these examples are illustrative only. Methods, kits, anddevices described herein may be used to detect and sequence a widevariety of nucleic acid sequences from a wide variety of samples,including, human, veterinary, industrial, and environmental.

Various embodiments disclosed herein use a self-contained nucleic acidanalysis pouch to assay a sample for the presence of various biologicalsubstances, illustratively antigens and nucleic acid sequences,illustratively in a single closed system. Such systems, includingpouches and instruments for use with the pouches, are disclosed in moredetail in U.S. Pat. Nos. 8,394,608; and 8,895,295; and U.S. PatentApplication No. 2014-0283945, herein incorporated by reference. However,it is understood that such pouches are illustrative only, and thenucleic acid preparation and amplification reactions discussed hereinmay be performed in any of a variety of open or closed system samplevessels as are known in the art, including 96-well plates, plates ofother configurations, arrays, carousels, and the like, using a varietyof nucleic acid purification and amplification systems, as are known inthe art.

While the terms “sample well”, “amplification well”, “amplificationcontainer”, or the like are used herein, these terms are meant toencompass wells, tubes, and various other reaction containers, as areused in these amplification systems. In one embodiment, the pouch isused to assay for multiple pathogens. The pouch may include one or moreblisters used as sample wells, illustratively in a closed system.Illustratively, various steps may be performed in the optionallydisposable pouch, including nucleic acid preparation, primary largevolume multiplex PCR, dilution of primary amplification product, andsecondary PCR, culminating with optional real-time detection orpost-amplification analysis such as melting-curve analysis. Further, itis understood that while the various steps may be performed in pouchesof the present invention, one or more of the steps may be omitted forcertain uses, and the pouch configuration may be altered accordingly.

As used herein, the term “point-of-use” refers to a step that isperformed by a system on a device or in a method either immediatelybefore or while the system is in use. For instance, drawing a vacuum onone of more chambers of an assay device at the “point-of-use” means thatthe vacuum is drawn shortly before (e.g., within an hour or less)performing an assay with the assay device or in situ during or afterperforming one or more steps of an assay with the assay device. Thiscontrasts with performing a step like drawing an appropriate vacuum atthe time of manufacture of the assay device and then storing the deviceunder appropriate vacuum until shortly before the time of use.

FIG. 1 shows an illustrative pouch 510 that may be used in variousembodiments, or may be reconfigured for various embodiments. Pouch 510is similar to FIG. 15 of U.S. Pat. No. 8,895,295, with like itemsnumbered the same. Fitment 590 is provided with entry channels 515 athrough 515 l, which also serve as reagent reservoirs or wastereservoirs. Illustratively, reagents may be freeze dried in fitment 590and rehydrated prior to use. Blisters 522, 544, 546, 548, 564, and 566,with their respective channels 514, 538, 543, 552, 553, 562, and 565 aresimilar to blisters of the same number of FIG. 15 of U.S. Pat. No.8,895,295. Second-stage reaction zone 580 of FIG. 1 is similar to thatof U.S. Pat. No. 8,895,295, but the second-stage wells 582 of highdensity array 581 are arranged in a somewhat different pattern. The morecircular pattern of high density array 581 of FIG. 1 eliminates wells incorners and may result in more uniform filling of second-stage wells582. As shown, the high density array 581 is provided with 102second-stage wells 582. Pouch 510 is suitable for use in the FilmArray®instrument (BioFire Diagnostics, LLC, Salt Lake City, Utah). However, itis understood that the pouch embodiment is illustrative only.

While other containers may be used, illustratively, pouch 510 may beformed of two layers of a flexible plastic film or other flexiblematerial such as polyester, polyethylene terephthalate (PET),polycarbonate, polypropylene, polymethylmethacrylate, mixtures,combinations, and layers thereof that can be made by any process knownin the art, including extrusion, plasma deposition, and lamination. Forinstance, each layer can be composed of one or more layers of materialof a single type or more than one type that are laminated together.Metal foils or plastics with aluminum lamination also may be used. Otherbarrier materials are known in the art that can be sealed together toform the blisters and channels. If plastic film is used, the layers maybe bonded together, illustratively by heat sealing. Illustratively, thematerial has low nucleic acid binding capacity.

For embodiments employing fluorescent monitoring, plastic films that areadequately low in absorbance and auto-fluorescence at the operativewavelengths are preferred. Such material could be identified by testingdifferent plastics, different plasticizers, and composite ratios, aswell as different thicknesses of the film. For plastics with aluminum orother foil lamination, the portion of the pouch that is to be read by afluorescence detection device can be left without the foil. For example,if fluorescence is monitored in second-stage wells 582 of thesecond-stage reaction zone 580 of pouch 510, then one or both layers atwells 582 would be left without the foil. In the example of PCR, filmlaminates composed of polyester (Mylar, DuPont, Wilmington Del.) ofabout 0.0048 inch (0.1219 mm) thick and polypropylene films of0.001-0.003 inch (0.025-0.076 mm) thick perform well. Illustratively,pouch 510 may be made of a clear material capable of transmittingapproximately 80%-90% of incident light.

In the illustrative embodiment, the materials are moved between blistersby the application of pressure, illustratively pneumatic pressure, uponthe blisters and channels. Accordingly, in embodiments employingpressure, the pouch material illustratively is flexible enough to allowthe pressure to have the desired effect. The term “flexible” is hereinused to describe a physical characteristic of the material of the pouch.The term “flexible” is herein defined as readily deformable by thelevels of pressure used herein without cracking, breaking, crazing, orthe like. For example, thin plastic sheets, such as Saran™ wrap andZiploc® bags, as well as thin metal foil, such as aluminum foil, areflexible. However, only certain regions of the blisters and channelsneed be flexible, even in embodiments employing pneumatic pressure.Further, only one side of the blisters and channels need to be flexible,as long as the blisters and channels are readily deformable. Otherregions of the pouch 510 may be made of a rigid material or may bereinforced with a rigid material. Thus, it is understood that when theterms “flexible pouch” or “flexible sample container” or the like areused, only portions of the pouch or sample container need be flexible.

Illustratively, a plastic film may be used for pouch 510. A sheet ofmetal, illustratively aluminum, or other suitable material, may bemilled or otherwise cut, to create a die having a pattern of raisedsurfaces. When fitted into a pneumatic press (illustratively A-5302-PDS,Janesville Tool Inc., Milton Wis.), illustratively regulated at anoperating temperature of 195° C., the pneumatic press works like aprinting press, melting the sealing surfaces of plastic film only wherethe die contacts the film. Likewise, the plastic film(s) used for pouch510 may be cut and welded together using a laser cutting and weldingdevice. Various components, such as PCR primers (illustratively spottedonto the film and dried), antigen binding substrates, magnetic beads,and zirconium silicate beads may be sealed inside various blisters asthe pouch 510 is formed. Reagents for sample processing can be spottedonto the film prior to sealing, either collectively or separately. Inone embodiment, nucleotide tri-phosphates (NTPs) are spotted onto thefilm separately from polymerase and primers, essentially eliminatingactivity of the polymerase until the reaction may be hydrated by anaqueous sample. If the aqueous sample has been heated prior tohydration, this creates the conditions for a true hot-start PCR andreduces or eliminates the need for expensive chemical hot-startcomponents. In another embodiment, components may be provided in powderor pill form and are placed into blisters prior to final sealing.

Pouch 510 may be used in a manner similar to that described in U.S. Pat.No. 8,895,295. In one illustrative embodiment, a 300 μl mixturecomprising the sample to be tested (100 μl) and lysis buffer (200 μl)may be injected into an injection port (not shown) in fitment 590 nearentry channel 515 a, and the sample mixture may be drawn into entrychannel 515 a. Water may also be injected into a second injection port(not shown) of the fitment 590 adjacent entry channel 515 l, and isdistributed via a channel (not shown) provided in fitment 590, therebyhydrating up to eleven different reagents, each of which were previouslyprovided in dry form at entry channels 515 b through 515 l. Illustrativemethods and devices for injecting sample and hydration fluid (e.g. wateror buffer) are disclosed in U.S. Patent Application No. 2014-0283945,herein incorporated by reference in its entirety, although it isunderstood that these methods and devices are illustrative only andother ways of introducing sample and hydration fluid into pouch 510 arewithin the scope of this disclosure. These reagents illustratively mayinclude freeze-dried PCR reagents, DNA extraction reagents, washsolutions, immunoassay reagents, or other chemical entities.Illustratively, the reagents are for nucleic acid extraction,first-stage multiplex PCR, dilution of the multiplex reaction, andpreparation of second-stage PCR reagents, as well as control reactions.In the embodiment shown in FIG. 1, all that need be injected is thesample solution in one injection port and water in the other injectionport. After injection, the two injection ports may be sealed. For moreinformation on various configurations of pouch 510 and fitment 590, seeU.S. Pat. No. 8,895,295, already incorporated by reference.

After injection, the sample may be moved from injection channel 515 a tolysis blister 522 via channel 514. Lysis blister 522 is provided withbeads or particles 534, such as ceramic beads or other abrasiveelements, and is configured for vortexing via impaction using rotatingblades or paddles provided within the FilmArray® instrument.Bead-milling, by shaking, vortexing, sonicating, and similar treatmentof the sample in the presence of lysing particles such as zirconiumsilicate (ZS) beads 534, is an effective method to form a lysate. It isunderstood that, as used herein, terms such as “lyse,” “lysing,” and“lysate” are not limited to rupturing cells, but that such terms includedisruption of non-cellular particles, such as viruses.

FIG. 4 shows a bead beating motor 819, comprising blades 821 that may bemounted on a first side 811 of support member 802, of instrument 800shown in FIG. 2. Blades may extend through slot 804 to contact pouch510. It is understood, however, that motor 819 may be mounted on otherstructures of instrument 800. In one illustrative embodiment, motor 819is a Mabuchi RC-280SA-2865 DC Motor (Chiba, Japan), mounted on supportmember 802. In one illustrative embodiment, the motor is turned at 5,000to 25,000 rpm, more illustratively 10,000 to 20,000 rpm, and still moreillustratively approximately 15,000 to 18,000 rpm. For the Mabuchimotor, it has been found that 7.2V provides sufficient rpm for lysis. Itis understood, however, that the actual speed may be somewhat slowerwhen the blades 821 are impacting pouch 510. Other voltages and speedsmay be used for lysis depending on the motor and paddles used.Optionally, controlled small volumes of air may be provided into thebladder 822 adjacent lysis blister 522. It has been found that in someembodiments, partially filling the adjacent bladder with one or moresmall volumes of air aids in positioning and supporting lysis blisterduring the lysis process. Alternatively, other structure, illustrativelya rigid or compliant gasket or other retaining structure around lysisblister 522, can be used to restrain pouch 510 during lysis. It is alsounderstood that motor 819 is illustrative only, and other devices may beused for milling, shaking, or vortexing the sample. In some embodiments,chemicals or heat may be used in addition to or instead of mechanicallysis.

Once the sample material has been adequately lysed, the sample is movedto a nucleic acid extraction zone, illustratively through channel 538,blister 544, and channel 543, to blister 546, where the sample is mixedwith a nucleic acid-binding substance, such as silica-coated magneticbeads 533. Alternatively, magnetic beads 533 may be rehydrated,illustratively using fluid provided from one of the entry channel 515c-515 e, and then moved through channel 543 to blister 544, and thenthrough channel 538 to blister 522. The mixture is allowed to incubatefor an appropriate length of time, illustratively approximately 10seconds to 10 minutes. A retractable magnet located within theinstrument adjacent blister 546 captures the magnetic beads 533 from thesolution, forming a pellet against the interior surface of blister 546.If incubation takes place in blister 522, multiple portions of thesolution may need to be moved to blister 546 for capture. The liquid isthen moved out of blister 546 and back through blister 544 and intoblister 522, which is now used as a waste receptacle. One or more washbuffers from one or more of injection channels 515 c to 515 e areprovided via blister 544 and channel 543 to blister 546. Optionally, themagnet is retracted and the magnetic beads 533 are washed by moving thebeads back and forth from blisters 544 and 546 via channel 543. Once themagnetic beads 533 are washed, the magnetic beads 533 are recaptured inblister 546 by activation of the magnet, and the wash solution is thenmoved to blister 522. This process may be repeated as necessary to washthe lysis buffer and sample debris from the nucleic acid-bindingmagnetic beads 533.

After washing, elution buffer stored at injection channel 515 f is movedto blister 548, and the magnet is retracted. The solution is cycledbetween blisters 546 and 548 via channel 552, breaking up the pellet ofmagnetic beads 533 in blister 546 and allowing the captured nucleicacids to dissociate from the beads and come into solution. The magnet isonce again activated, capturing the magnetic beads 533 in blister 546,and the eluted nucleic acid solution is moved into blister 548.

First-stage PCR master mix from injection channel 515 g is mixed withthe nucleic acid sample in blister 548. Optionally, the mixture is mixedby forcing the mixture between 548 and 564 via channel 553. Afterseveral cycles of mixing, the solution is contained in blister 564,where a pellet of first-stage PCR primers is provided, at least one setof primers for each target, and first-stage multiplex PCR is performed.If RNA targets are present, an RT step may be performed prior to orsimultaneously with the first-stage multiplex PCR. First-stage multiplexPCR temperature cycling in the FilmArray® instrument is illustrativelyperformed for 15-20 cycles, although other levels of amplification maybe desirable, depending on the requirements of the specific application.The first-stage PCR master mix may be any of various master mixes, asare known in the art. In one illustrative example, the first-stage PCRmaster mix may be any of the chemistries disclosed in U.S. Pat. No.9,932,634, herein incorporated by reference, for use with PCR protocolstaking 20 seconds or less per cycle.

After first-stage PCR has proceeded for the desired number of cycles,the sample may be diluted, illustratively by forcing most of the sampleback into blister 548, leaving only a small amount in blister 564, andadding second-stage PCR master mix from injection channel 515 i.Alternatively, a dilution buffer from 515 i may be moved to blister 566then mixed with the amplified sample in blister 564 by moving the fluidsback and forth between blisters 564 and 566. If desired, dilution may berepeated several times, using dilution buffer from injection channels515 j and 515 k, or injection channel 515 k may be reserved,illustratively, for sequencing or for other post-PCR analysis, and thenadding second-stage PCR master mix from injection channel 515 h to someor all of the diluted amplified sample. It is understood that the levelof dilution may be adjusted by altering the number of dilution steps orby altering the percentage of the sample discarded prior to mixing withthe dilution buffer or second-stage PCR master mix comprising componentsfor amplification, illustratively a polymerase, dNTPs, and a suitablebuffer, although other components may be suitable, particularly fornon-PCR amplification methods. If desired, this mixture of the sampleand second-stage PCR master mix may be pre-heated in blister 564 priorto movement to second-stage wells 582 for second-stage amplification.Such preheating may obviate the need for a hot-start component(antibody, chemical, or otherwise) in the second-stage PCR mixture.

The illustrative second-stage PCR master mix is incomplete, lackingprimer pairs, and each of the 102 second-stage wells 582 is pre-loadedwith a specific PCR primer pair. If desired, second-stage PCR master mixmay lack other reaction components, and these components may bepre-loaded in the second-stage wells 582 as well. Each primer pair maybe similar to or identical to a first-stage PCR primer pair or may benested within the first-stage primer pair. Movement of the sample fromblister 564 to the second-stage wells 582 completes the PCR reactionmixture. Once high density array 581 is filled, the individualsecond-stage reactions are sealed in their respective second-stageblisters by any number of means, as is known in the art. Illustrativeways of filling and sealing the high density array 581 withoutcross-contamination are discussed in U.S. Pat. No. 8,895,295, alreadyincorporated by reference. Illustratively, the various reactions inwells 582 of high density array 581 are simultaneously or individuallythermal cycled, illustratively with one or more Peltier devices,although other means for thermal cycling are known in the art.

In certain embodiments, second-stage PCR master mix contains the dsDNAbinding dye LCGreen® Plus (BioFire Diagnostics, LLC) to generate asignal indicative of amplification. However, it is understood that thisdye is illustrative only, and that other signals may be used, includingother dsDNA binding dyes and probes that are labeled fluorescently,radioactively, chemiluminescently, enzymatically, or the like, as areknown in the art. Alternatively, wells 582 of array 581 may be providedwithout a signal, with results reported through subsequent processing.

When pneumatic pressure is used to move materials within pouch 510, inone embodiment, a “bladder” may be employed. The bladder assembly 810, aportion of which is shown in FIGS. 2-3, includes a bladder plate 824housing a plurality of inflatable bladders 822, 844, 846, 848, 864, and866, each of which may be individually inflatable, illustratively by acompressed gas source. Because the bladder assembly 810 may be subjectedto compressed gas and used multiple times, the bladder assembly 810 maybe made from tougher or thicker material than the pouch. Alternatively,bladders 822, 844, 846, 848, 864, and 866 may be formed from a series ofplates fastened together with gaskets, seals, valves, and pistons. Otherarrangements are within the scope of this invention. Alternatively, anarray or mechanical actuators and seals may be used to seal channels anddirect movement of fluids between blisters. A system of mechanical sealsand actuators that may be adapted for the instruments described hereinis described in detail in WO 2018/022971, the entirety of which isincorporated herein by reference.

Success of the secondary PCR reactions is dependent upon templategenerated by the multiplex first-stage reaction. Typically, PCR isperformed using DNA of high purity. Methods such as phenol extraction orcommercial DNA extraction kits provide DNA of high purity. Samplesprocessed through the pouch 510 may require accommodations be made tocompensate for a less pure preparation. PCR may be inhibited bycomponents of biological samples, which is a potential obstacle.Illustratively, hot-start PCR, higher concentration of Taq polymeraseenzyme, adjustments in MgCl₂ concentration, adjustments in primerconcentration, and addition of adjuvants (such as DMSO, TMSO, orglycerol) optionally may be used to compensate for lower nucleic acidpurity. While purity issues are likely to be more of a concern withfirst-stage amplification, it is understood that similar adjustments maybe provided in the second-stage amplification as well.

When pouch 510 is placed within the instrument 800, the bladder assembly810 is pressed against one face of the pouch 510, so that if aparticular bladder is inflated, the pressure will force the liquid outof the corresponding blister in the pouch 510. In addition to bladderscorresponding to many of the blisters of pouch 510, the bladder assembly810 may have additional pneumatic actuators, such as bladders orpneumatically-driven pistons, corresponding to various channels of pouch510. FIGS. 2-3 show an illustrative plurality of pistons or hard seals838, 843, 852, 853, and 865 that correspond to channels 538, 543, 553,and 565 of pouch 510, as well as seals 871, 872, 873, 874 that minimizebackflow into fitment 590. When activated, hard seals 838, 843, 852,853, and 865 form pinch valves to pinch off and close the correspondingchannels. To confine liquid within a particular blister of pouch 510,the hard seals are activated over the channels leading to and from theblister, such that the actuators function as pinch valves to pinch thechannels shut. Illustratively, to mix two volumes of liquid in differentblisters, the pinch valve actuator sealing the connecting channel isactivated, and the pneumatic bladders over the blisters are alternatelypressurized, forcing the liquid back and forth through the channelconnecting the blisters to mix the liquid therein. The pinch valveactuators may be of various shapes and sizes and may be configured topinch off more than one channel at a time. While pneumatic actuators arediscussed herein, it is understood that other ways of providing pressureto the pouch are contemplated, including various electromechanicalactuators such as linear stepper motors, motor-driven cams, rigidpaddles driven by pneumatic, hydraulic or electromagnetic forces,rollers, rocker-arms, and in some cases, cocked springs. In addition,there are a variety of methods of reversibly or irreversibly closingchannels in addition to applying pressure normal to the axis of thechannel. These include kinking the bag across the channel, heat-sealing,rolling an actuator, and a variety of physical valves sealed into thechannel such as butterfly valves and ball valves. Additionally, smallPeltier devices or other temperature regulators may be placed adjacentthe channels and set at a temperature sufficient to freeze the fluid,effectively forming a seal. Also, while the design of FIG. 1 is adaptedfor an automated instrument featuring actuator elements positioned overeach of the blisters and channels, it is also contemplated that theactuators could remain stationary, and the pouch 510 could betransitioned such that a small number of actuators could be used forseveral of the processing stations including sample disruption,nucleic-acid capture, first and second-stage PCR, and processingstations for other applications of the pouch 510 such as immuno-assayand immuno-PCR. Rollers acting on channels and blisters could proveparticularly useful in a configuration in which the pouch 510 istranslated between stations. Thus, while pneumatic actuators are used inthe presently disclosed embodiments, when the term “pneumatic actuator”is used herein, it is understood that other actuators and other ways ofproviding pressure may be used, depending on the configuration of thepouch and the instrument.

Turning back to FIG. 2, each pneumatic actuator is connected tocompressed air source 895 via valves 899. While only several hoses 878are shown in FIG. 2, it is understood that each pneumatic fitting isconnected via a hose 878 to the compressed gas source 895. Compressedgas source 895 may be a compressor, or, alternatively, compressed gassource 895 may be a compressed gas cylinder, such as a carbon dioxidecylinder. Compressed gas cylinders are particularly useful ifportability is desired. Other sources of compressed gas are within thescope of this invention. Similar pneumatic control may be provided inthe embodiments of FIGS. 12-16, for control of fluids in pouch 1400, orother actuators, servos, or the like may be provided.

Several other components of instrument 810 are also connected tocompressed gas source 895. A magnet 850, which is mounted on a secondside 814 of support member 802, is illustratively deployed and retractedusing gas from compressed gas source 895 via hose 878, although othermethods of moving magnet 850 are known in the art. Magnet 850 sits inrecess 851 in support member 802. It is understood that recess 851 canbe a passageway through support member 802, so that magnet 850 cancontact blister 546 of pouch 510. However, depending on the material ofsupport member 802, it is understood that recess 851 need not extend allthe way through support member 802, as long as when magnet 850 isdeployed, magnet 850 is close enough to provide a sufficient magneticfield at blister 546, and when magnet 850 is fully retracted, magnet 850does not significantly affect any magnetic beads 533 present in blister546. While reference is made to retracting magnet 850, it is understoodthat an electromagnet may be used and the electromagnet may be activatedand inactivated by controlling flow of electricity through theelectromagnet. Thus, while this specification discusses withdrawing orretracting the magnet, it is understood that these terms are broadenough to incorporate other ways of withdrawing the magnetic field. Itis understood that the pneumatic connections may be pneumatic hoses orpneumatic air manifolds, thus reducing the number of hoses or valvesrequired. It is understood that similar magnets and methods foractivating the magnets may be used in the embodiments of FIGS. 12-16.

The various pneumatic pistons 868 of pneumatic piston array 869 are alsoconnected to compressed gas source 895 via hoses 878. While only twohoses 878 are shown connecting pneumatic pistons 868 to compressed gassource 895, it is understood that each of the pneumatic pistons 868 areconnected to compressed gas source 895. Twelve pneumatic pistons 868 areshown.

A pair of temperature control elements are mounted on a second side 814of support 802. As used herein, the term “temperature control element”refers to a device that adds heat to or removes heat from a sample.Illustrative examples of a temperature control element include, but arenot limited to, heaters, coolers, Peltier devices, resistance heaters,induction heaters, electromagnetic heaters, thin film heaters, printedelement heaters, positive temperature coefficient heaters, andcombinations thereof. A temperature control element may include multipleheaters, coolers, Peltiers, etc. In one aspect, a given temperaturecontrol element may include more than one type of heater or cooler. Forinstance, an illustrative example of a temperature control element mayinclude a Peltier device with a separate resistive heater applied to thetop and/or the bottom face of the Peltier. While the term “heater” isused throughout the specification, it is understood that othertemperature control elements may be used to adjust the temperature ofthe sample.

As discussed above, first-stage heater 886 may be positioned to heat andcool the contents of blister 564 for first-stage PCR. As seen in FIG. 2,second-stage heater 888 may be positioned to heat and cool the contentsof second-stage blisters 582 of array 581 of pouch 510, for second-stagePCR. It is understood, however, that these heaters could also be usedfor other heating purposes, and that other heaters may be included, asappropriate for the particular application.

As discussed above, while Peltier devices, which thermocycle between twoor more temperatures, are effective for PCR, it may be desirable in someembodiments to maintain heaters at a constant temperature.Illustratively, this can be used to reduce run time, by eliminating timeneeded to transition the heater temperature beyond the time needed totransition the sample temperature. Also, such an arrangement can improvethe electrical efficiency of the system as it is only necessary tothermally cycle the smaller sample and sample vessel, not the muchlarger (more thermal mass) Peltier devices. For instance, an instrumentmay include multiple heaters (i.e., two or more) at temperatures setfor, for example, annealing, elongation, denaturation that arepositioned relative to the pouch to accomplish thermal cycling. Twoheaters may be sufficient for many applications. In various embodiments,the heaters can be moved, the pouch can be moved, or fluids can be movedrelative to the heaters to accomplish thermal cycling. Illustratively,the heaters may be arranged linearly, in a circular arrangement, or thelike. Types of suitable heaters have been discussed above, withreference to first-stage PCR.

When fluorescent detection is desired, an optical array 890 may beprovided. As shown in FIG. 2, optical array 890 includes a light source898, illustratively a filtered LED light source, filtered white light,or laser illumination, and a camera 896. Camera 896 illustratively has aplurality of photodetectors each corresponding to a second-stage well582 in pouch 510. Alternatively, camera 896 may take images that containall of the second-stage wells 582, and the image may be divided intoseparate fields corresponding to each of the second-stage wells 582.Depending on the configuration, optical array 890 may be stationary, oroptical array 890 may be placed on movers attached to one or more motorsand moved to obtain signals from each individual second-stage well 582.It is understood that other arrangements are possible. The embodimentfor second-stage heaters shown in FIG. 18 provides the heaters on theopposite side of pouch 510 from that shown in FIG. 2. Such orientationis illustrative only and may be determined by spatial constraints withinthe instrument. Provided that second-stage reaction zone 580 is providedin an optically transparent material, photodetectors and heaters may beon either side of array 581.

As shown, a computer 894 controls valves 899 of compressed air source895, and thus controls all of the pneumatics of instrument 800. Inaddition, many of the pneumatic systems in the instrument may bereplaced with mechanical actuators, pressure applying means, and thelike in other embodiments. Computer 894 also controls heaters 886 and888, and optical array 890. Each of these components is connectedelectrically, illustratively via cables 891, although other physical orwireless connections are within the scope of this invention. It isunderstood that computer 894 may be housed within instrument 800 or maybe external to instrument 800. Further, computer 894 may includebuilt-in circuit boards that control some or all of the components, andmay also include an external computer, such as a desktop or laptop PC,to receive and display data from the optical array. An interface,illustratively a keyboard interface, may be provided including keys forinputting information and variables such as temperatures, cycle times,etc. Illustratively, a display 892 is also provided. Display 892 may bean LED, LCD, or other such display, for example.

Other prior art instruments teach PCR within a sealed flexiblecontainer. See, e.g., U.S. Pat. Nos. 6,645,758, 6,780,617, and9,586,208, herein incorporated by reference. However, including the celllysis within the sealed PCR vessel can improve ease of use and safety,particularly if the sample to be tested may contain a biohazard. In theembodiments illustrated herein, the waste from cell lysis, as well asthat from all other steps, remains within the sealed pouch. Still, it isunderstood that the pouch contents could be removed for further testing.

FIG. 2 shows an illustrative instrument 800 that could be used withpouch 510. Instrument 800 includes a support member 802 that could forma wall of a casing or be mounted within a casing. Instrument 800 mayalso include a second support member (not shown) that is optionallymovable with respect to support member 802, to allow insertion andwithdrawal of pouch 510. Illustratively, a lid may cover pouch 510 oncepouch 510 has been inserted into instrument 800. In another embodiment,both support members may be fixed, with pouch 510 held into place byother mechanical means or by pneumatic pressure.

In the illustrative example, heaters 886 and 888 are mounted on supportmember 802. However, it is understood that this arrangement isillustrative only and that other arrangements are possible. Illustrativeheaters include Peltiers and other block heaters, resistance heaters,electromagnetic heaters, and thin film heaters, as are known in the art,to thermocycle the contents of blister 864 and second-stage reactionzone 580. Bladder plate 810, with bladders 822, 844, 846, 848, 864, 866,hard seals 838, 843, 852, 853, and seals 871, 872, 873, 874 form bladderassembly 808, which may illustratively be mounted on a moveable supportstructure that may be moved toward pouch 510, such that the pneumaticactuators are placed in contact with pouch 510. When pouch 510 isinserted into instrument 800 and the movable support member is movedtoward support member 802, the various blisters of pouch 510 are in aposition adjacent to the various bladders of bladder assembly 810 andthe various seals of assembly 808, such that activation of the pneumaticactuators may force liquid from one or more of the blisters of pouch 510or may form pinch valves with one or more channels of pouch 510. Therelationship between the blisters and channels of pouch 510 and thebladders and seals of assembly 808 is illustrated in more detail in FIG.3.

FIG. 5A shows another illustrative embodiment of a pouch 5000 (alsoreferred to herein as a ‘science card’) that may be used in variousembodiments, or may be reconfigured for various embodiments describedherein for PCR, microbial testing, or for a variety of other tests. Thepouch 5000 may be configured for use in an instrument described in WO2017/147085, herein incorporated by reference, or in a variety of otherinstruments. The illustrative pouch 5000 of FIG. 5A includes a number ofzones or blisters where sample preparation, nucleic acid amplification,and detection can occur. The illustrative pouch 5000 may include asample preparation blister 5005 where a sample containing nucleic acidsto be amplified and analyzed may be introduced into the pouch 5000, afirst-stage PCR blister 5010, a volumetric dilution well 5015 formeasuring a portion of the product from first-stage PCR prior tosecond-stage PCR, and a second-stage PCR array 5081 that includes anumber of individual reaction wells 5082. The volumetric well 5015 mayalso be fluidly coupled to blisters 5020 and 5025, where reagents forsecond-stage PCR may be introduced and mixed with the contents of thedilution well 5015. In one example, a sample for second-stage PCR may beprepared by repeatedly mixing the contents of volumetric well 5015 withreagents for second-stage PCR between blisters 5020 and 5025. Thesecond-stage array 5081 may also be fluidly connected to a wastereceptacle 5035. Alternatively, blister 5010 may be used for both samplepreparation and first-stage PCR and blister 5005 may be used as a wastereceptacle for, for example, sample preparation waste(s).

Blisters 5005, 5010, 5020, and 5025, dilution well 5015, andsecond-stage array 5081 may be fluidly connected by channels 5050 a-5050e. Sample and reagent may be entered into the pouch 5000 via entrychannels 5040 a-5040 f and entry ports 5045 a-5045 f. Alternatively,pouch 5000 may be fitted with a device similar in form to fitment 590 ofFIG. 1 for introduction of sample and reagents into the pouch 5000. Inaddition, the pouch 5000 may include dehydrated (e.g., freeze dried)reagents in a fitment or a similar structure that may be hydrated with asuitable hydration buffer prior to use of the pouch. In yet anotherembodiment, liquid reagents may be provided in pouch 5000.

In one embodiment, the pouch 5000 may be fabricated from a number oflayers of material (layers of the same material or layers of differenttypes of material) that are sealed together to form the pouch 5000. InFIGS. 5B and 5C, cutaway views are shown along the lines B-B and C-Cillustrating layers of material that may be used to fabricate differentparts of the pouch 5000. In one region of the pouch 5000 illustrated inFIG. 5B, the illustrative pouch 5000 may be fabricated from a firstlayer of film 5090 that is bonded to a second layer of film 5098. Layers5090 and 5098 may be bonded together by any conventional means known inthe art such as, but not limited to, heat and pressure, sonic welding,or laser welding. FIG. 5B also illustrates that a blister or a channel(e.g., channel 5040 c) may be formed in the pouch 5000 by leaving anopen area between the film layers 5090 and 5098 and defining theboundaries of the open area with sealed margins along the opening—anillustrative weld is shown at 5084 in FIGS. 5A and 5B. FIG. 5Cillustrates another region of the pouch 5000 that includes a thick cardmaterial that may be used to form the wells of the second-stage array5081. This region of the illustrative pouch 5000 may be fabricated froma first film layer 5090, a pressure sensitive adhesive layer 5092, acard layer 5094, a second pressure sensitive adhesive layer 5096, and asecond film layer 5098. In one illustrative example, the wells 5082 ofthe second-stage array 5081 may be formed in the card layer 5094. In analternative to forming the channels (e.g., channel 5040 c) and blisters(e.g., blister 5005) by leaving open space between the film layers(e.g., film layers 5090 and 5098) as illustrated in FIG. 5B, the card5094 layer could be extended and the blisters and/or the channels couldbe formed by making appropriate cutouts in the card layer 5094.Likewise, channels 5050 a-5050 e and entry channels 5040 a-5040 f may beformed by making appropriate cutouts in the either the first or secondpressure sensitive adhesive layers 5092 and 5096. One will appreciatethat other configurations are possible. It is understood that while theillustrative blister areas are flexible, the card layer 5094 optionallymay be less flexible and may be rigid, and still be part of a flexiblesample container. Thus, it is understood that a “flexible pouch” needonly be flexible in certain zones. Alternatively or in addition, flowchannels between the blister areas can be formed by adding another filmlayer, tubing, or rigid layer above film layer 5090 or below film layer5098 and welding the layers together, leaving open blister areas andchannels between the layers.

While other materials may be used, illustratively, the film layers ofpouch 5000 may be formed from a flexible plastic film or other flexiblematerial similar to the pouch 510 described in FIG. 1. For instance,pouch 5000 may be fabricated from materials such as, but not limited to,such as polyester, polyethylene terephthalate (PET), polycarbonate,polypropylene (PP), polymethylmethacrylate, combinations, mixtures, andlaminated layers thereof that can be made by any process known in theart, including extrusion, plasma deposition, and lamination. Similarmaterials (e.g., polycarbonate) may be used for the card layer 5094.Other materials, including metal foils or plastics with aluminumlamination, may also be used. Other barrier materials are known in theart that can be sealed together to form the blisters and channels. Ifplastic film is used, the layers may be bonded together, illustrativelyby heat sealing or laser welding. Illustratively, the material has lownucleic acid binding capacity. If fluorescence detection is used,optically transparent material may be used in the appropriate areas ofthe pouch (e.g., in the vicinity of the second-stage array).

In addition to or in lieu of the foregoing examples of film materials, abarrier film may be used in one or more of the layers used to form pouch5000 or any of the other pouches described herein. For instance, barrierfilms may be desirable for some applications because they have low watervapor and/or oxygen transmission rates that may be lower thanconventional plastic films. For example, typical barrier films havewater vapor transmission rates (WVTR) in a range of about 0.01 g/m²/24hrs to about 3 g/m²/24 hrs, preferably in a range of about 0.05 g/m²/24hrs to about 2 g/m²/24 hrs (e.g., no more than about 1 g/m²/24 hrs) andoxygen transmission rates in a range of about 0.01 cc/m²/24 hrs to about2 cc/m²/24 hrs, preferably in a range of about 0.05 cc/m²/24 hrs toabout 2 cc/m²/24 hrs (e.g., no more than about 1 cc/m²/24 hrs). Examplesof barrier films include, but are not limited to, films that can bemetallized by vapor deposition of a metal (e.g., aluminum or anothermetal) or sputter coated with an oxide (e.g., Al₂O₃ or SiO_(x)) oranother chemical composition. A common example of a metallized film isaluminized Mylar, which is metal coated biaxially oriented PET (BoPET).In some applications, coated barrier films can be laminated with a layerof polyethylene, PP, or a similar thermoplastic, which providessealability and improves puncture resistance. As with conventionalplastic films, barrier films layers used to fabricate a pouch may bebonded together, illustratively by heat sealing. Illustratively, thematerial has low nucleic acid binding and low protein binding capacity.Other barrier materials are known in the art that can be sealed togetherto form the blisters and channels.

Pouch 5000 may be used in a manner similar to that described above forpouch 510 and/or in a manner similar to that described in U.S. Pat. No.8,895,295. Referring again to FIG. 5A, two alternative sequences forfilling the pouch, preparing a sample, performing first-stage PCR, andperforming second-stage PCR are described. In a first example method,sample preparation and first-stage PCR may be performed in separateblisters. This is referred to herein as the “three zone method,” withthe three zones being sample preparation, first-stage PCR, andsecond-stage PCR. In the following examples describing the “three zonemethod” and the “two-zone method,” one will appreciate that pouch 5000is one embodiment of a pouch and that other pouch configurations may beadapted to the three- and/or two-zone methods.

In a first step, a sample is injected into blister 5005 via fill channel5040 a. In one embodiment, cells, viruses, and the like may be lysed inblister 5005 using the wiping system described in detail elsewhereherein. Alternatively, cell lysis may be accomplished with analternative lysis device such as, but not limited to, a sonicationdevice or a bead beater or by chemical lysis. Optionally, lysis may beaided by heating the sample (e.g., to about 70-90° C.) with one or moreheater elements of the heater assembly described in detail elsewhereherein. Following lysis, the sample may be cooled with a thermoelectriccooler element (i.e., a Peltier element) to a temperature in a range ofabout 0° C. to about 20° C. (e.g., about 10-15° C.) to aid in nucleicacid recovery with, for example, silica-coated magnetic beads. Othercooler elements include, but are not limited to, fluid or gas heatexchange elements, fan cooled heat sinks, heat pipes, condensationunits, and the like.

Magnetic beads may be injected into blister 5005 via fill channel 5040 aor 5040 b for use in recovering nucleic acids from the lysate.Alternatively, cells to be lysed, lysis beads, magnetic beads, lysisbuffer, and the like may be injected together or sequentially intoblister 5005 prior to lysis. Illustratively, the magnetic beads and thelysate may be mixed cold (e.g., in a range of about 0-10° C.,illustratively by adjusting the temperature of one of the heaters). Oncethe magnetic beads and the lysate have been thoroughly mixed for asufficient time, the magnetic beads may be gathered in blister 5005 witha magnet illustratively provided in the instrument and the spent lysatemay be sent to liquid waste via channel 5040 b. Then wash buffer may beinjected via fill channel 5040 a. The wash buffer and the magnetic beadsmay be mixed cold (e.g., in a range of about 0-10° C.). The magneticbeads may be gathered again and the spent wash buffer may be flushed toliquid waste via channel 5040 b. The wash cycle may be repeated at leastone more time. Following the wash, an elution buffer (optionallyincluding first-stage PCR primers) may be injected into blister 5005 viafill channel 5040 a. The elution buffer (plus first-stage PCR primers)and the magnetic beads optionally may be mixed hot (e.g., at about70-90° C.), illustratively, under control of one or more heaters.

For first-stage PCR, PCR master mix (e.g., a polymerase, dNTPs, andother amplification components known in the art) may be injected intoblister 5010 via fill channel 5040 c. The PCR master mix may be heated(e.g., to about 57° C.) prior to introduction of the eluate from themagnetic beads. In blister 5005, the magnetic beads may be gatheredagain and the eluate may be sent to blister 5010 via channel 5050 a.

In one embodiment, first-stage PCR may be performed in blister 5010 withrotary movement of a wiper system illustrated in WO 2017/147085, alreadyincorporated by reference, illustratively under temperature control oftwo heaters. Alternatively, first-stage PCR thermocycling may beperformed by translating a heater assembly or the pouch 5000 so thatblister 5010 may be under control of one heater and then another of aheater assembly that includes two different heaters at two differenttemperatures (e.g., at an annealing and at a denaturation temperature).The channels into and out of blister 5010 may be closed, illustrativelywith seals similar to those described in reference to FIGS. 2 and 3,during first stage PCR. In some embodiments, it may be possible to speedup first-stage PCR in the pouch by employing a volume reductionprotocol. For instance, a volume reduction protocol may includeperforming several cycles (e.g., 1-10) of PCR with an initial volume(e.g., ˜100 μL) in blister 5010, purging approximately half the volumeof blister 5010, performing several more cycles of PCR (e.g., 5-10), andagain purging approximately half the volume of blister 5010. Volumereduction can reduce the cycle time for a PCR reaction because smallervolumes of liquid have less thermal mass and can be thermocycled morequickly than larger volumes.

Following a sufficient number of cycles of first-stage PCR (e.g., 20-30cycles), a small sample (e.g., ˜1-5 μL) of first-stage PCR may be sentto dilution well 5015 via channel 5050 b; channels 5050 c-5050 e may beclosed. The sample for second-stage PCR may be prepared by injecting asecond-stage PCR master mix into blister 5025 via channel 5040 e. Sealschannels 5050 b and 5050 e may be closed, seals 5050 c and 5050 d may beopened and the sample in well 5015 may be mixed with the master mix bymixing between blisters 5025 and 5020 and well 5015 to dilutefirst-stage PCR product for second-stage PCR. Blisters 5020 and 5025 andwell 5015 may be heated prior to or during mixing for a physical“hot-start.” Channel 5050 e is then opened and seals 5050 c and 5050 dmay be closed so that the second-stage PCR mix can be transferred intothe second-stage PCR array 5081. In another embodiment, the pouch 5000may include one or more additional dilution wells and sets of mixingblisters downstream from well 5015 and blisters 5025 and 5020 andupstream from array 5081. For example, in some embodiments withconcentrated first-stage PCR primers or with highly concentratedproduct, it may be desirable to dilute the first-stage primers andproduct to a degree greater than can be achieved with one dilution well.Thermocycling for second-stage PCR in array 5081 may illustratively beaccomplished by translating the heater assembly back and forth asdescribed in detail elsewhere herein.

In the second exemplary method, sample preparation and first-stage PCRmay be performed in the same blister. This is referred to herein as the“two zone method,” wherein sample preparation and first-stage PCR areperformed in one zone and second-stage PCR is performed in a secondzone. In a first step, a sample may be injected into blister 5010 viafill channel 5040 c. In one embodiment, cells, viruses, and the like arelysed in blister 5010 using the wiping system described in detailelsewhere herein. Alternatively, cell lysis may be accomplished with analternative lysis device such as, but not limited to, a sonicationdevice or a bead beater or chemical lysis. Lysis may be aided by heatingthe sample to an elevated temperature (e.g., about 70-90° C.) with oneor more heater elements of the heater assembly described in detailelsewhere herein. Following lysis, the sample may optionally be cooledwith a thermoelectric cooler element (i.e., a Peltier element) to areduced temperature (e.g., a temperature below ambient temperature suchas, but not limited to, ˜0-10° C.).

Magnetic beads may be injected into blister 5010 via fill channel 5040 cin order to recover nucleic acids from the lysate. In one embodiment,the magnetic beads and the lysate may be mixed cold (e.g., at atemperature in a range of about 0-10° C.) after lysis. In anotherembodiment, a combination of cells to be lysed, lysis buffer, magneticbeads, and, optionally, lysis beads may be injected together intoblister 5010 such that lysis and nucleic acid capture may occur atsubstantially the same time. Once the magnetic beads and the lysate havebeen thoroughly mixed for a sufficient time, the magnetic beads may begathered in blister 5010 with a magnet and the spent lysate may be sentto blister 5005 (i.e., the liquid waste blister in this example) liquidwaste via channel 5050 a. Then wash buffer may be injected into blister5010 via fill channel 5040 c. Optionally, the wash buffer and themagnetic beads may be mixed cold (e.g., at a temperature in a range ofabout 0-10° C.). The magnetic beads are gathered again and the spentwash buffer may be flushed to blister 5005. The wash cycle may berepeated one or more times, if desired. After wash, nucleic acids may beeluted from the beads (optionally at an elevated temperature of, e.g.,about 70-90° C.) by injecting an elution buffer (plus first-stage PCRprimers) into blister 5010. The magnetic beads and any remaining lysisbeads (if present) may be collected into the upstream half of blister5010, and sent to waste blister 5005 via channel 5050 a.

For first-stage PCR, the wiper system may be set and first-stage PCRmaster mix may be injected into channel 5040 d and optionally held at anelevated temperature (e.g., about 57° C.) if a true hot-start may bedesired. First-stage PCR master mix may be mixed with primers andtemplate in blister 5010 and first-stage PCR may be performed asdescribed above.

Following first-stage PCR, the protocol may proceed to second-stage PCRas described above for the “three zone method.”

When fluorescent detection is desired, an optical array may be provided.An optical array may include a light source, illustratively a filteredLED light source, filtered white light, or illumination, and a camera.The camera illustratively has a plurality of photodetectors eachcorresponding to a second-stage well in array 5081 of pouch 5000.Alternatively, the camera may take images that contain all of thesecond-stage wells, and the image may be divided into separate fieldscorresponding to each of the second-stage wells. Depending on theconfiguration, the optical array may be stationary, or the optical arraymay be placed on movers attached to one or more motors and moved toobtain signals from each individual second-stage well. It is understoodthat other arrangements are possible.

Referring now to FIG. 6A, an array 6000 of wells that may be used forsecond-stage PCR is illustrated in greater detail. Array 6000 may be astandalone array or it may be included as a group of wells in a largerarray, such as part of array 5081. Array 6000 includes individual wells6002, 6004, 6006, 6008, and 6010. Each of wells 6002, 6004, 6006, 6008,and 6010 may be used for a second-stage PCR reaction. In the illustratedembodiment, the wells 6002, 6004, 6006, 6008, and 6010 are fluidlyconnected to a fill channel 6012; holes 6014, 6016, 6018, 6020, and 6022are formed in the fill channel 6012 for filling each of the wells. Inone embodiment, wells of a second-stage array (e.g., wells 6002, 6004,6006, 6008, and 6010) may be under a partial vacuum to facilitatedrawing fluid from the fill channel 6012 into the wells. In oneembodiment, the wells 6002, 6004, 6006, 6008, and 6010 can be sealed offfrom the fill channel 6012 and from each other and leakage or mixing offluid between wells, which may be referred to as “cross-talk,” can beminimized or prevented by applying a seal (e.g., a heat seal) orpressure in or around the region illustrated at 6024. Thus, the singleseal may be applied in the region indicated at 6024 to close off wells6002, 6004, 6006, 6008, and 6010 from fill channel 6012 and from eachother to prevent well-to-well cross-talk. The cross-sectional structureof the array 6000 and the flow path for filling the wells is illustratedbelow in FIGS. 6B and 6C. And while array 6000 is illustrated with fivewells 6002, 6004, 6006, 6008, and 6010 associated with the fill channel6012, one will appreciate that more or fewer reaction wells can beassociated with a fill channel and that multiple fill channels can befluidly connected to multiple clusters of wells. Multiple arrays 6000may be used in combination to create larger arrays.

Referring now to FIGS. 6B and 6C, FIG. 6B is a cross-sectional viewillustrated along the line B-B of FIG. 6A, and FIG. 6C is an alternativeembodiment showing different embodiments of well filling systems. Theportion of the array 6000 illustrated in cross section in FIGS. 6B and6C is made of layers similar to those shown in FIG. 5C; it should benoted that the array 6000 may be included as part of the pouch 5000shown in FIG. 5A instead of array 5081. The well 6006 b shown in FIG. 6Bmay be defined by a first film layer 6030 b, a second film layer 6032 b,an adhesive layer 6034 b, a card layer 6036 b in which well 6006 b maybe defined, a second adhesive layer 6038 b, and a third (outside) filmlayer 6040 b. The well 6006 c shown in FIG. 6C is quite similar and maybe defined by a first film layer 6030 c, a second film layer 6032 c, anadhesive layer 6034 c, a card layer 6036 c in which a well 6006 c may beformed, a second adhesive layer 6038 c, and a third (outside) film layer6040 c. The key differences between 6B and 6C are in how the wells 6006b and 6006 c are formed in the surrounding layers and in how the wellsmay be filled.

In FIG. 6B, the fill channel 6012 b may be formed by leaving a gapbetween the first and second film layers 6030 b and 6032 b where liquidcan flow. FIG. 6C shows a similar fill channel 6012 c formed by leavinga gap between the first and second film layers 6030 c and 6032 c. Thefill channels may be defined by weld lines 6026 b or 6026 c that sealthe first and second film layers together around the array. An exampleof how these welds may be applied is shown in FIG. 6A at weld region6026 that includes an outer weld 6026 a and an inner weld 6026 bdefining the fill channel 6012 and the space around the wells. In FIG.6B, the fill hole 6018 b may be formed by making selective cutouts inthe second film layer 6032 b and in the first adhesive layer 6034 b. InFIG. 6C, the fill hole 6018 c may be formed by making a selective cutoutin the second film layer 6032 c that is adjacent to a correspondingcutout in the first adhesive layer 6034 c.

In FIG. 6B, a well filling channel that flows around and over the well6006 b for filling the well may be formed by making a cutout 6042 b inthe card layer 6036 b and a cutout 6044 b in the second adhesive layer6038 b, although other ways of forming these channels are possible. Thedesign of the well filling channel of FIG. 6B may, for instance, help tosuppress cross-talk between wells because the flow path is convoluted.Likewise, because the fill channel 6012 b and the fill hole 6018 b areformed between two film layers 6030 b and 6032 b, the fill hole 6018 band access to the well 6006 b can be sealed, illustratively with a heatseal device or by pressure, illustratively by a bladder that inflates inthe region adjacent to 6024 of FIG. 6A or against all or part of thearray 6000. In FIG. 6C, the well filling channel 6018 c flows directlyinto the well 6006 c and may be formed by making a cutout 6046 c in thefirst adhesive layer 6034 c that fluidly connects the fill hole 6018 cto the well 6006 c. It is expected that the filling design of FIG. 6Cwill also generally suppress cross-talk between wells. However, thedesign of FIG. 6C may be sealed, illustratively, with a heat sealdevice, which may provide better sealing than pressure alone.

In some embodiments, the wells of the second-stage array may be under atleast a partial vacuum to facilitate filling of the wells with fluid forsecond stage PCR. Generally, this may mean that the pouch is storedunder a partial vacuum from the time of manufacture until packagingsurrounding the sample vessel is opened at the time of use. In oneillustrative example, FIGS. 7A and 7B illustrate an embodiment of asecond-stage array that may be used as an alternative to vacuum storagewhile still allowing reliable well filling in the second-stage array. Aswill be discussed in detail below, FIGS. 7A and 7B schematicallyillustrate an array that includes a vacuum way that may, illustratively,be formed in a liquid filling channel that allows a partial vacuum to bepulled on the array in situ.

FIGS. 7A and 7B illustrate another embodiment of a second-stage array11000 that that may be filled without having the pouch manufactured andstored under partial vacuum. Second-stage array 11000 is similar tosecond-stage array 6000 illustrated in FIG. 6A. Second-stage array11000, which is defined in part by weld line 11026, includessecond-stage wells 11002, 11004, 11006, 11008, and 11010 that can eachbe provided with a unique second-stage primer pair and that can befilled with component for second-stage PCR (e.g., diluted product fromfirst-stage PCR, polymerase, dNTPs, etc.) and be thermal cycled forsecond-stage analysis, as described in detail elsewhere herein. Thesecond-stage wells are fillable from fill channel 11012, which is influid communication with fill holes 11014, 11016, 11028, 11020, and11022 and fluidic vias 11052 a-11052 e that are associated with eachsecond-stage well. Integrally formed in the fill channel and in fluidcommunication with each of the fill holes, vias, and second-stage wellsis an illustrative vacuum way 11050. Vacuum way 11050 is in turn influid communication with port 11051 that may be placed on a portion ofthe pouch away from the array. In one embodiment, port 11051 may includea hole 11051 a that provides fluid access to the vacuum way.Illustratively, hole 11051 a may be formed in either layer 11030 or11032. Vacuum way 11050 can illustratively be used to pull a partialvacuum on the second stage wells in situ (e.g., by a vacuum pump in theinstrument during a pouch run) so that the pouch does not need to bemanufactured or stored under vacuum. In one embodiment, vacuum channel11050 may illustratively be connected to a remote vacuum hub on thepouch that can be connected to a vacuum source.

Referring now to FIG. 7B, a cross-section of the fill channel 11012 andthe vacuum way 11050 is illustrated. In the illustrated embodiment, thefill channel 11012 is formed as an open space between two film layers11030 and 11032 that are joined together (e.g., laser welded) on theiredges at 11026. Illustratively, the vacuum way 11050 may be formed as asub-channel in one of layers 11030 or 11032. In the illustratedembodiment, the vacuum way 11050 has an arch shape that is designed tohold channel 11012 open and connect the fill holes, vias, andsecond-stage wells to the vacuum source via vacuum way 11050 and port11051. Without the vacuum channel 11050, channel 11012 may tend tocollapse or “kiss” shut when a vacuum in pulled on the channel andprevent evacuation of the wells. In one embodiment, the vacuum channel11050 may be formed as a recessed conduit in one of the layers with aheat forming fixture (e.g., an appropriately shaped hot ‘debossing’wire). In other embodiments, the vacuum channel 11050 may be formed bylaser etching, xurography, or the like. As illustrated in FIG. 7B,vacuum way 11050, which is an example of a heat formed channel, has anarch shape that supports the plastic and holds the channel open so thatthe vacuum can draw air out of the second-stage wells. While an archshape is illustrated, it is understood that channels having other shapesmay be used herein that are used to maintain the fill channel in an openposition. Nonetheless, the vacuum way 11050 can be sealed by, forexample, bonding layers 11030 and 11032 to one another by applying aheat seal over the vacuum way 11050, illustratively, near port 11051 andaway from the array wells.

In one embodiment, a vacuum of at least 1-150 millibar (e.g., 2-10millibar or, more preferably, 2-5 millibar) may be pulled on the secondstage array (or portion of another reaction container configured forhaving a vacuum drawn thereon in situ) for 10-120 seconds in situ (e.g.,in an instrument configured for drawing a vacuum on one or more portionsof a reaction container and performing reactions un the reactioncontainer, or immediately prior to inserting the reaction container intoan instrument for running an analytical method). Following pulling avacuum, the vacuum channel may be sealed (e.g., heat seated) and thevacuum may be released at port 11051, which leaves the wells of thearray under a partial vacuum. Experiments on prototype arrays withvacuum channels similar to what is described above have shown thatpulling a vacuum in situ can be as effective as manufacturing andstoring the pouch under vacuum.

Some embodiments of the present disclosure can include an array assemblythat comprises a plurality of wells arranged in an array, an integrallyformed (e.g., in-molded) channel system in fluid communication with aplurality of wells, and an integrally formed (e.g., in-molded) vacuumport in fluid communication with the channel system. The channel systemcan also have an integrally formed fluid opening separate from thevacuum port. In some embodiments, a fluid reservoir or source with anopenable seal (e.g., with a frangible seal, a peelable seal, or otherseals as are known in the art) may be positioned adjacent to the arrayassembly. In some embodiments, a vacuum can be applied to the arrayassembly by drawing a vacuum at the vacuum port; the openable sealbetween the fluid source and the array assembly may typically be keptclosed while evacuating the array assembly. By opening the sealseparating the fluid source from the array assembly, a fluid sample canbe drawn by the vacuum through the channel system and into the pluralityof wells.

FIG. 8A depicts an illustrative array assembly 8000 according to anembodiment of the present disclosure that may be used as part of anypouch or card discussed herein or in other such embodiments. As depictedin FIG. 8A, array assembly 8000 comprises a card or card layer 8002having a plurality of wells 8004 arranged in an array 8001. Asillustrated, for example, in reference to FIGS. 8C and 8D, card 8002 maybe disposed in a sealed reaction container between two or more filmlayers. Illustrative card 8002 comprises a first (lower) surface 8016, asecond (upper) surface 8018 opposite first surface 8016, and a perimeteredge 8020 extending therebetween. Card 8002 can be any suitable size,including thickness, length, width, etc. For example, card 8002 may havea thickness of about 0.2-0.3 mm and the wells may be sized so that thecard thickness and the well diameter yield wells of 0.20-0.5 μl,illustratively 0.3 μl. As depicted in FIG. 8A, illustrative card 8002has a rectangular shape, with optionally rounded corners. In otherembodiments, however, card 8002 can have any suitable shape. Wells 8004may be arranged in rows, as shown in FIG. 8A, in circular or hexagonalarrangements, as in FIGS. 1 and 6A, or in other arrangements. It isunderstood that the term “array” includes any such arrangement of wells.

Wells 8004 can comprise, be formed of, or be defined by a hole oraperture extending through card 8002 (e.g., from first surface 8016 tosecond surface 8018). In alternative embodiments, the hole or openingcan extend only part way through card 8002. Thus, in some embodiments,wells 8004 can comprise, be formed of, or be defined by a recess orindentation in card 8002 or second surface 8018 thereof. The recess orindentation forming wells 8004 can have any suitable depth within (orless than) the thickness of card 8002 (between first surface 8016 andsecond surface 8018). As depicted in FIG. 8A, wells 8004 has a round orcylindrical configuration. In alternative embodiments, however, wells8004 can have other shapes or configurations, some of which are shown inFIGS. 12A-12D.

As discussed above, array 8001 can have any suitable configuration. Asdepicted in the illustrative example of FIG. 8A, array 8001 comprises aplurality of wells 8004 disposed in a plurality of rows 8022.Illustratively, wells 8004 a, 8004 b, and 8004 c are in a first row 8022a of wells 8004. A second row 8022 b of wells 8004 is adjacent to firstrow 8022 a of wells 8004. A third row 8022 c of wells 8004 is adjacentto second row 8022 b, opposite first row 8022 a. Additional rows 8022 ofwells 8004 can be adjacent to, aligned (parallel) with, and/or disposedbetween or opposite first row 8022 a, second row 8022 b, and/or thirdrow 8022 c. Accordingly, illustrative rows 8022 of wells 8004 arearranged in a grid or grid-like configuration (e.g., with wells 8004 ofeach row 8022 aligned or substantially aligned with wells 8004 ofanother row 8022).

As depicted in FIG. 8A, array 8001 of wells 8004 comprises ten rows 8022of ten wells 8004. In alternative embodiments, however, array 8001 canhave any suitable number of rows 8022, such as, for example, greaterthan 100 rows, from 1 to 100 rows, 2 to 80 rows, 3 to 75 rows, 4 to 60rows, 5 to 50 rows, 6 to 40 rows, 7 to 20 rows, 8 to 12 rows, or anysuitable number of rows, or range of number of rows disposedtherebetween. In some embodiments, array 8001 can have at least 1 row, 2rows, 3 rows, 4 rows, 5 rows, 6 rows, 8 rows, 10 rows, 12 rows, 16 rows,24 rows, 32 rows, 48 rows, 64 rows, or 96 rows.

In some embodiments, row(s) 8022 can have any suitable number of wells8004, such as, for example, greater than 100 wells, from 1 to 100 wells,2 to 80 wells, 3 to 75 wells, 4 to 60 wells, 5 to 50 wells, 6 to 40wells, 7 to 20 wells, 8 to 12 wells, or any suitable number of wells, orrange of number of wells disposed therebetween. In some embodiments,row(s) 8022 can have at least 1 well, 2 wells, 3 wells, 4 wells, 5wells, 6 wells, 8 wells, 10 wells, 12 wells, 16 wells, 24 wells, 32wells, 48 wells, 64 wells, or 96 wells. In at least one embodiment,array 8001 can have at least one row 8022 of a plurality of wells 8004,a plurality of rows 8022 of at least one well 8004, or a plurality ofrows 8022 of a plurality of wells 8004.

Array assembly 8000 further comprises a channel system 8006 in fluidcommunication with the plurality of wells 8002, with a fluid entryopening 8010 in a first side 8012 of card 8002, and with a vacuum port8008 in a second side 8014 of card 8002. Accordingly, as describedabove, fluid opening 8010 can be separate from vacuum port 8008. In theillustrated embodiment, vacuum port 8008 is disposed on an opposite sideof wells 8004 from fluid opening 8010. It will be appreciated, however,that this orientation is illustrative only, and other configurations arepossible. As depicted in FIGS. 8C-D, it is understood that arrayassembly 8000 may be incorporated into a pouch or science card, whereinfluid opening 8010 is in fluid communication with other reaction zones.

As depicted in FIG. 8A, vacuum port 8008 comprises a hole 8008 a in card8002. Hole 8008 a extends through card 8002, from first surface 8016 tosecond surface 8018. Similar to wells 8004, in an alternativeembodiment, hole 8008 a can extend only part way through card 8002.Thus, in some embodiments, hole 8008 a can comprise, be formed of, or bedefined by a recess or indentation in card 8002 or second surface 8018thereof. The recess or indentation forming hole 8008 a can have anysuitable depth within (or less than) the thickness of card 8002 (betweenfirst surface 8016 and second surface 8018). As depicted in FIG. 8A,hole 8008 a has a round or cylindrical configuration. However, hole 8008a can alternatively have another shape or configuration. In analternative embodiment, vacuum port 8008 can comprise an end or otherportion of channel system 8006.

As depicted in FIG. 8A, channel system 8006 comprises a manifold channelassembly 8024 in fluid communication with fluid entry channel 8034.Manifold channel assembly 8024 comprises a first main channel 8026 thatis fluidly connected to fluid entry channel 8034, a second main channel8028 that is fluidly connected to vacuum port 8008, and a plurality ofbranch channels 8030 extending between first main channel 8026 andsecond main channel 8028. Each branch channel 8030 extends alongside oneor more rows 8022 of wells 8004. Illustratively, a first branch channel8030 a extends alongside first row 8022 a, a second branch channel 8030b extends alongside second row 8022 b, a third branch channel 8030 cextends alongside third row 8022 c, and so forth. A plurality ofconnection channels 8032 extend from each branch channel 8030 torespective wells 8004 in each row 8022. Illustratively, each connectionchannel 8032 extends from one well 8004 in a particular row 8022 to abranch channel 8030 extending alongside that particular row 8022.Illustratively, each of the wells 8004 in a particular row 8022 is influid communication with the branch channel 8030 extending alongsidethat particular row 8022 by means of a respective connection channel8032.

FIG. 8B is a detailed view of card 8002 at well 8004 a, connectionchannel 8032, and branch channel 8030 a. As illustrated in FIG. 8B,connection channel 8032 and branch channel 8030 a (and, indeed, all ofchannel system 8006) can comprise, be formed of, or be defined by arecess or indentation in card 8002 or second surface 8018 thereof. Therecess or indentation forming channel system 8006 or one or morecomponents thereof can have any suitable depth within (or less than) thethickness of card 8002 (between first surface 8016 and second surface8018). As depicted in FIG. 8A, channel system 8006 has a squaredconfiguration. In alternative embodiments, however, channel system 8006can have other shapes or configurations. In some embodiments, certaincomponents of channel system 8006 can have a depth greater than or lessthan other components of channel system 8006. As depicted in FIG. 8A,however, all components of channel system 8006 have the same depth andwells 8004 have a greater depth.

Referring now to FIGS. 12A-12D, examples of various well filling pathsfor wells in an array are shown. FIGS. 8A-8C showed one example of a“convoluted” array well filling path that includes wells 8004, arrayflow channels 8030, and straight well fill channels 8032 as the wellfilling path. FIGS. 12A-12D illustrate examples of well filling pathsthat are less direct than well fill channels 8032. Each example in FIGS.12A-12D includes a well 1200 a-1200 d and an array flow channel 1230a-1230 d. FIG. 12A illustrates an embodiment of a spiral channel 1232 aleading into well 1204 a. FIG. 12B illustrates a switchback channel 1232b leading into well 1204 b. FIG. 12C illustrates a channel 1232 c thatincludes a spiral portion 1208 c and a switch back portion 1210 cleading into well 1204 c. FIG. 12D illustrates a channel 1232 d with aspiral switchback portion 1208 d and a parallel switchback portion 1210d leading into well 1204 d. When placed in a pouch, the wells and thechannels illustrated in FIGS. 8A-8C and 12A-12D would be sealed with atleast one layer on the top and bottom. Thus, the only flow paths intothe wells is through the flow channels and the well channels. While somepaths are more “convoluted” than others, the combination of the flowchannels and the well channels generally isolates the wells from oneanother and minimizes mixing (i.e., cross-talk) between wells as thewells are filled. The foregoing examples are merely illustrativeexamples of convoluted well filling paths and one will appreciate thatother well filling paths may be used.

Referring again to FIG. 8A, illustrative channel system 8006 alsoincludes a fluid access channel 8034 extending from fluid opening 8010to first main channel 8026. As depicted in FIG. 8A, fluid opening 8010can be disposed in perimeter edge 8020 and/or second upper surface 8018of card 8002. Fluid access channel 8034 can be configured to introduceor permit a fluid into channel system 8006 or manifold channel assembly8024 thereof. For instance, as described further herein, card 8002 canbe sealed in a pouch or between two layers of film (see, e.g., FIGS. 8Cand 8D). Accordingly, in some embodiments, access channel 8034 can beused to fluidly transfer a liquid sample from the pouch into array 8001or wells 8004 thereof. In some embodiments, fluid access channel 8034can be in fluid communication with a fluid source (see, e.g., FIGS. 8Cand 8D). In at least one embodiment, the fluid source with which accesschannel 8034 is in fluid communication with a blister in a film pouch(see e.g., blister 566 of pouch 510 of FIG. 1). An optional fluidchannel (see e.g., channel 565 of FIG. 1) can extend between accesschannel 8034 and the fluid source. Other suitable fluid sources as knownin the art are also contemplated herein.

Channel system 8006 also includes a vacuum port channel 8036 extendingfrom vacuum port 8008 to second main channel 8028. Accordingly, eachwell 8004 is in fluid communication with a connection channel 8032,which is in fluid communication with a branch channel 8030, which is influid communication with first main channel 8026 and second main channel8028. First main channel 8026 is in fluid communication with fluidaccess channel 8034, which is in fluid communication with fluid opening8010. Accordingly, each well 8004 is in fluid communication with fluidopening 8010 (by means of channel system 8006) from at least twodirections (either (i) directly through first main channel 8026, its ownbranch channel 8030, and its own connection channel 8032, or (ii)indirectly through first main channel 8026, a connection channel 8032from a different row, second main channel 8028, its own branch channel8030, and its own connection channel 8032). This parallel arrangementwith multiple connection channels extending between two or more mainchannels allows for pulling a vacuum on all wells and for filling allwells even if one of the channels becomes obstructed. Second mainchannel 8028 is in fluid communication with port channel 8036, which isin fluid communication with vacuum port 8008. Accordingly, each well8004 is in fluid communication with vacuum port 8008 (by means ofchannel system 8006). It is understood that this arrangement isillustrative, and many alternative arrangements are possible.

Card 8002 can also have one or more optional alignment indicators 8038.As shown in FIG. 8A, optional alignment indicator(s) 8038 comprise anopening or a hole extending into or through one or more of surfaces ofcard layer 8002. Other optional alignment indicator(s) 8038 include arecess (or indentation) or a marking (e.g., printed pattern or image)disposed on one or more surfaces of card layer 8002.

In some embodiments, one or more of the features or components of card8002 (e.g., wells 8004, parts of channel system 8006, port hole 8008 a,alignment indicators 8038, etc.) can be formed during manufacture,illustratively during injection molding, of card 8002. Alternatively,one or more of the features or components of card 8002 can be formed byremoving material of card 8002.

FIG. 8C depicts an illustrative embodiment of an array assembly 8000 a.Array assembly 8000 a comprises card (or card layer) 8002 disposed in apouch 8040. Pouch 8040 can comprise or be formed of a first layer and asecond layer. Illustrative card layer 8002 is disposed (e.g.,sandwiched) between first film layer 8042 and second film layer 8044. Insome embodiments, film layers 8042 and 8044 can be bonded to the top andbottom surfaces 8016 and 8018 of card layer 8002. Film layers 8042 and8044 can also be selectively bonded to each other to create, forexample, fluid channels and blisters between the film layers.Accordingly, some embodiments of the present disclosure relate to anarray assembly 8000 a comprising a card layer 8002 having the pluralityof wells 8004 formed therein, the first film layer 8042 bonded to afirst side or surface 8016 of card layer 8002, and/or a second filmlayer 8044 bonded to a second side or surface 8018 of card layer 8002.While film layers are used in this and other embodiments, it isunderstood that other materials may be used consistent with theapplication of array assembly 8000 a.

As depicted in FIG. 8D, pouch 8040 can be pre-formed (e.g., prior todisposing card 8002 therein in some embodiments. Illustratively,(portions of) first film layer 8042 and second film layer 8044 can bebonded together, such as by thermoforming (e.g., laser welding or heatwelding), by hot rolling (e.g., by partially bonding the first andsecond film layers 8042 and 8044 together by rolling the layers betweenhot rollers during manufacture), or by an adhesive (e.g., temperaturesensitive adhesive, pressure sensitive adhesive, etc.). For instance,pouch 8040 includes a first bonded portion 8056 a, a second bondedportion 8056 b, a third bonded portion 8056 c. Bonded portions 8056 a,8056 b, and 8056 c can at least partially bound or form a fluidreservoir (or blister) 8046, a fluid channel 8048 in fluid communicationwith blister 8046, and a pocket 8050 in fluid communication with fluidchannel 8048. In some embodiments, while bonded portions 8056 a, 8056 b,and 8056 c of pouch 8040, or first film layer 8042 and second film layer8044 thereof, can be (permanently) bonded together (e.g. such as by theheat welding or pressure sensitive adhesive), blister 8046, fluidchannel 8048, and pocket 8050 can be left unbonded, or openably (i.e.,reversibly) bonded together. In some embodiments, the unbonded portionscan be defined by one or more bond lines or weld lines 8052.

Illustratively, second film layer 8044 can be laid on top of first filmlayer 8042 to form a stack or layup. One or more portions of theoverlaid film layers or stack can then be bonded together at bondedportions 8056 a, 8056 b, and 8056 c, such as by thermoforming (e.g.,heat welding) or by an adhesive (e.g., temperature sensitive adhesive,pressure sensitive adhesive, etc.). For example, one or more heatedpressure plates or other selective bonding member bearing the pattern ofbonded portions 8056 a, 8056 b, and 8056 c can be used to form pouch8040 from stack film layers 8042 and 8044 by applying heat and a force(e.g., pressure) to one or more sides of the layup.

Illustratively, as discussed above with reference to pouch 510, filmlayers 8042 and 8044 may be formed of plastic or other material that canbe softened and/or (partially) melted such that the layers or surfacesthereof become bonded (e.g., thermoformed or heat-welded) together. Forexample, first and second film layers 8042 and 8044 can comprise or beformed of one or more materials with similar melting temperature(s)(Tm), glass transition temperature(s) (Tg), etc. Illustratively, firstand second film layers 8042 and 8044 can comprise or be formed of one ormore polymeric (e.g., plastic) or other materials, includingcombinations or blends thereof. Accordingly, a layup or stack of firstfilm layer 8042 and second film layer 8044 can heated to at least the Tmor Tg of one or more of the materials (e.g., the material having thehighest Tm or Tg), such that the layers or surfaces thereof becomebonded (e.g., heat-welded) together (e.g., with application of a force(e.g., pressure) to the heated or melted layup.

In one or more alternative embodiments, an adhesive layer bearing thepattern of bonded portions 8056 a, 8056 b, and 8056 c can be disposedbetween film layers 8042 and 8044 in the stack or layup. Illustratively,the adhesive layer can be or comprise a pressure sensitive adhesive(PSA). One or more pressure plates or other selective bonding memberbearing the pattern of bonded portions 8056 a, 8056 b, and 8056 c can beused to form pouch 8040 from stack film layers 8042 and 8044 by applyinga force (e.g., pressure) to one or more sides of the layup.Alternatively, a temperature sensitive adhesive or other adhesive asknown in the art can be used to form pouch 8040.

As further depicted in FIG. 8D, pocket 8050 can have an unbonded, openend or opening 8054 (e.g., between bond lines 8052 of bonded portions8056 b and 8056 c, respectively) and one or more optional alignmentindicators 8038 a. Optional alignment indicator(s) 8038 a can comprisean opening or hole through one or more of film layers 8042 and 8044.Alternatively, optional alignment indicator(s) 8038 a can comprise amarking (e.g., printed pattern or image) disposed on one or more of filmlayers 8042 and 8044. Pouch 8040, or one or more of film layers 8042 and8044 thereof, can include a piercing 8008 b, in some embodiments.

Returning now to FIG. 8C, and with the continued reference to FIG. 8D,card layer 8002 can be inserted through opening 8054 into pouch 8050.Optional alignment indicator(s) 8038 of card layer 8002 can be alignedwith optional alignment indicator(s) 8038 a of first film layer 8042and/or second film layer 8044, thereby indicating a proper alignmentand/or position of card layer 8002 within pocket 8050.

Film layers 8042 and 8044 can be bound to inserted card layer 8002, suchas by thermoforming or heat welding. Illustratively, one or more heatedpressure plates or other bonding members can be used to bond film layers8042 and 8044 to card layer 8002. The heated pressure plate(s) can havea substantially flat and/or uniform bonding surface in some embodiments.Alternatively, the heated pressure plate(s) can have a bonding surfacebearing the pattern of card layer 8002 or components thereof.

It will also be appreciated that one or more adhesive layers bearing thepattern of card layer 8002 or components thereof (see FIG. 8A), can(also or alternatively) be disposed on one or more of surfaces 8016 and8018, or corresponding surfaces of film layers 8042 and 8044. Asillustrated in FIG. 5C, for example, a card layer (5094) can be bondedto a lower film layer (5098) by means of a first adhesive layer (5096),and to an upper film layer (5090) by means of a second adhesive layer(5092). Similarly, returning to FIG. 8C, first (lower) surface 8016 ofcard layer 8002 can be bonded to first film layer 8042 by means of afirst adhesive layer (not shown). Likewise, second (upper) surface 8018of card layer 8002 can be bonded to second film layer 8044 by means of asecond adhesive layer (not shown). The first and/or second adhesivelayers can be or comprise one or more pressure sensitive adhesives,temperature sensitive adhesives, or other adhesives, as known in the artand/or described herein.

A fourth bonded portion 8056 d can then be formed in a manner similar tothose described above, thereby sealing opening 8054 in a closedconfiguration, indicated at closed end 8054 a. Thus, card layer 8002 canbe (or become) bonded to pouch 8040 or film layers 8042 and 8044thereof, and sealed within pocket 8050. In some embodiments, an unbondedpocket portion 8050 a can be disposed at one or more sides of card layer8002 within sealed pouch 8040. As depicted in FIG. 8C, for instance,unbonded pocket portion 8050 a surrounds card layer 8002 inside sealedpouch 8040. Unbonded pocket portion 8050 a can be defined by bond lines(e.g., weld lines) 8052 of bonded portions 8056 a, 8056 b, 8056 c, and8056 d. Similarly, a fluid channel 8048 and reservoir (or blister) 8046can be defined by bond lines 8052 of bonded portions 8056 a.

In an alternative embodiment, array assembly 8000 a can be formed withcard layer 8002 disposed between unbonded sheets 8042 and 8044.Illustratively, a layup or stack of card layer 8002 disposed betweenfirst film layer 8042 and second film layer 8044 can be bonded asdescribed above and/or with the pattern of pouch 8040 or bond lines 8052thereof. For example, first and second film layers 8042 and 8044 andcard layer 8002 can comprise or be formed of one or more (respective)materials with similar melting temperature(s) (Tm), glass transitiontemperature(s) (Tg), etc. Illustratively, first and second film layers8042 and 8044 and card layer 8002 can comprise or be formed of one ormore polymeric (e.g., plastic) or other materials, includingcombinations or blends thereof. In a preferred embodiment, first andsecond film layers 8042 and 8044 and card layer 8002 can comprise or beformed of polypropylene or a blend thereof. One will appreciate,however, that the layers can also be formed of other materials as knownin the art.

The layup or stack of card layer 8002 disposed between first film layer8042 and second film layer 8044 can heated to at least the Tm or Tg ofone or more of the materials (e.g., the material having the highest Tmor Tg). A force (e.g., pressure) can also be applied to the layup, suchthat the layers or surfaces thereof become bonded (e.g., heat-welded)together. Alternatively, as described previously, one or more adhesivelayers (e.g., bearing the pattern of card layer 8002 and/or bond lines8052 of pouch 8040) can be disposed between card layer 8002 and one ormore of first film layer 8042 and second film layer 8044, such that cardlayer 8002 becomes bonded to first film layer 8042 and/or second filmlayer 8044 by means of the adhesive layer(s).

As further illustrated in FIG. 8C, a fluid or liquid (sample) 8062 canbe disposed in fluid reservoir (or blister) 8046. In the illustratedembodiment, the fluid 8062 can disposed (e.g., injected) into blister8046 prior to insertion of card layer 8002 into pocket 8050. In otherembodiments, such as those depicted in the corresponding drawings (seee.g., FIGS. 1 and 3), one or more additional fluid channels can beconnected to and/or in fluid communication with a fluid reservoir (orblister). Accordingly, it will be appreciated that one or moreadditional fluid channels 8048 can extend from blister 8046, such thatfluid 8062 can be introduced into blister 8046 after the formation ofpouch 8040.

An openable seal can also be formed pouch 8040, such as in area 8060 offluid channel 8048. As described previously, the openable seal can be orcomprise a frangible or an openable seal (e.g., a peelable seal) formedby the electrostatic, tactile, or other attraction between film layers8042 and 8044, rather than by bonding through thermoforming or adhesive.The openable seal can be configured to retain fluid 8062 within blister8046 (e.g., such that fluid 8062 does not become inadvertently and/orprematurely disposed in pocket 8050).

Array assembly 8000 a, or pouch 8040 thereof, can include at least oneopening or piercing 8008 b. Illustratively, piercing(s) 8008 b can beformed in one or more of film layers 8042 and 8044 during manufacture ofpouch 8040 or film layers 8042 and 8044 thereof. Alternatively,piercing(s) 8008 b can be formed (in array assembly 8000 a) during anillustrative analytical method. For instance, piercing(s) 8008 b can beformed by a piercing element of analytical device (not shown) prior to avacuum being applied at vacuum port 8008. Illustratively, piercing 8008b can be substantially aligned with hole 8008 a of card layer 8002. Insome embodiments, piercing 8008 b and hole 8008 a can form vacuum port8008. In other words, vacuum port 8008 can comprise piercing 8008 b andhole 8008 a in some embodiments.

With reference to FIGS. 8A and 8B, by (openably) sealing channel system8006 (e.g., at or near fluid opening 8010, such as at area 8060 in theadjacent fluid channel 8048, illustratively with a pressure seal or anopenable seal (e.g., a frangible seal or a peelable seal) between thefilm layers 8042 and 8044 that form the fluid channel 8048), arrayassembly 8000, 8000 a can be evacuated by applying a vacuum at vacuumport 8008 by means of a vacuum device—e.g., a piston pump (not shown).The seal at area 8060 allows the applied vacuum to evacuate port channel8036, second main channel 8028, each branch channel 8030, first mainchannel 8026, fluid access channel 8034, each connection channel 8032,and each well 8004 of array 8001. In this context, channel system 8006comprises or constitutes a vacuum channel. Illustratively, since vacuumis applied to the channel system prior to filling array assembly 8000,8000 a (e.g., with fluid sample 8062), the fluid sample (or first-stageamplicon in a two-step PCR system) is not introduced into the vacuumdevice, thereby minimizing or preventing contamination from connectionwith the vacuum device.

With array assembly 8000 evacuated and the applied vacuum maintained(e.g., such as by sealing off vacuum port 8008 at area 8064 of portchannel 8036, illustratively by pressure and/or heat sealing), the sealat area 8060 can be opened, drawing fluid into array assembly 8000(i.e., through fluid opening 8010, through first main channel 8026,through branch channels 8030, through connection channels 8032, and intoarray 8001 and wells 8004 thereof). By sealing port channel 8036 at area8064, fluid is also drawn into second main channel 8028 and a portion ofport channel 8036 (up to the seal). In this context, channel system 8006comprises or constitutes an array fill channel. Accordingly, channelsystem 8006 comprises or constitutes an array fill channel (or channelassembly), as well as a vacuum channel (or channel assembly).

In the illustrated embodiment, each well 8004 can be isolated from allother wells 8004 by sealing branch channels 8030 between connectionchannels 8032. In at least one embodiment, a plurality of seals canextend across one or more (e.g., all) of the branch channels 8030between one or more (e.g., all) of the connection channels 8032. Becauseeach of the wells 8004 in a particular row 8022 communicates with anadjacent branch channel 8030 through a particular connection channel8032, sealing across branch channels 8030 (e.g., perpendicular to thelength of branch channels 8030 and rows 8022 of wells 8004) betweenconnection channels 8032 substantially reduces or eliminates cross-talkbetween wells 8004.

By using (a manifold-type) channel system 8006 as both a vacuum channeland an array fill channel, card 8002 can be kept relatively small. Itwill be appreciated, however, that in some embodiments, array assembly8000 can comprise a vacuum channel and a separate array fill channel.For instance, in an alternative embodiment, array assembly 8000 cancomprise a dual manifold configuration, in which branch channels 8030are not in fluid communication with both first main channel 8026 andsecond main channel 8028. Instead, a first set of branched (fill)channels 8030 can extend from first main channel 8026 and a second setof branched (vacuum) channels 8030 can extend from second main channel8028. Respective connection channels 8032 can extend from each branchchannel 8030 to respective wells 8004. A first connection channel 8032can fluidically connect a particular well 8004 to an adjacent branchedvacuum channel 8030 on a first side of the well 8004, well a secondconnection channel 8032 can fluidically connect the particular well 8004to an adjacent branched fill channel 8030 on a second side of the well8004.

As described above, vacuum port 8008 need not be disposed on an oppositeside of wells 8004 from fluid opening 8010. For instance, vacuum port8008 can be disposed on the same or adjacent side of wells 8004 as fluidopening 8010. Illustratively, vacuum port 8008 and fluid opening 8010can be disposed on opposite ends of the same or adjacent side of wells8004 as. Vacuum port 8008 can even be disposed adjacent to fluid opening8010. For instance, in an alternative embodiment, channel system 8006,or manifold channel assembly 8024 thereof, can have only one mainchannel 8026. Illustratively, port channel 8036 can extend from mainchannel 8026, such that vacuum port 8008 or hole 8008 a thereof can bein fluid communication with main channel 8026. In such a configuration,a fluid channel in fluid communication with access channel 8034 canstill be openably sealed (e.g., adjacent to fluid opening 8010), and avacuum cans still be applied through vacuum port 8008.

FIGS. 9A and 9B show an illustrative embodiment of second-stage 9580using a barrier layer. Sandwiched between first layer 9518 and secondlayer 9519 of pouch 9510 is high density array 9581, with wells 9582. Asbest seen in FIG. 9B, pierced layer 9585, with piercings 9586, isprovided on one side of high density array 9581 to act as the physicalbarrier to minimize cross-talk between wells 9582 upon filling of array9581, and a second layer 9587 is provided on the opposite side of highdensity array 9581 to form the bottom of wells 9582. In the illustratedembodiment, second-stage array 9580 is fabricated with pierced layer9585 bonded to first layer 9518. In layer 9518, features are formed by,for example, bonding selected areas and impressing features into filmlayer 9518 to form a fluid flow channel 9053 and a concurrent vacuumchannel 9050 to allow the pulling of a vacuum on the array in situ(i.e., at point of use) as an alternative to manufacturing and storingthe array under vacuum. Illustratively, layer 9518 includes a fluid fillchannel 9053 formed by bonding (e.g., by laser welding) layer 9518 tolayer 9585. Fluid fill channel 9053, which is defined by laser weldlines 9052, is formed so as to be in fluid communication with each ofthe pierced holes 9586 in the layer 9585. Vacuum way 9050 is concurrentwith and formed in fluid fill channel 9053 and is also in fluidcommunication with each of the pierced holes 9586 in the layer 9585. Inone embodiment, vacuum way 9050 may be heat formed by shaping the filmof film layer 9518 with a hot wire or the like to impress a channel intothe film. Illustratively, pierced layer 9585 and first layer 9518 arebonded (e.g., welded) to one another around their edges at 9026. Piercedlayer 9585 is bonded to high density array layer 9581, e.g., by andadhesive layer and/or heat sealing, and first layer 9518 is bonded(e.g., laser welded) to pierced layer 9585 at several weld lines 9052parallel to and between columns of pierced layer holes 9586 to definethe fill channel 9053. Fluid fill channel 9053, pierced layer 9585 andopenings 9586, and array wells 9582 may be in fluid communication withan upstream fluid well via fluid channel opening 9565.

FIG. 9C illustrates a cross section along the line C-C of the layers ofthe array 9580. While FIG. 9A is shown in exploded view, FIG. 9Cillustrates the relationship of the pouch film and array layers, thevacuum way 9050, and the fluid flow channel 9053. The “stack” of thesecond-stage amplification zone 9580 includes a first pouch film layer9518 (i.e., a first outer layer of the pouch), a pierced layer 9585, anarray 9581, a second layer 9587 (i.e., the array backing layer), and asecond pouch film layer 9519 (i.e., a second outer pouch film layer).The cross-section of FIG. 9C illustrates one piercing 9586 and one well9582 and how they relate to the vacuum way 9050, the fluid flow channel9053, and the welds 9052 and 9026. While one piercing 9586 and one well9582 are shown for ease and clarity, one can see from FIGS. 9A and 9Cthat the vacuum way 9050 and the fluid flow channel 9053 are fluidlyconnected to all of the piercings 9586 and wells 9582 of thesecond-stage amplification zone 9580.

In the illustrated embodiment, the fluid flow channel 9053 is formed asan open space between first layer 9518 and pierced layer 9585 that arejoined together (e.g., laser welded) at welds 9052 and 9026 that definethe fluid flow channel 9053. Illustratively, the vacuum way 9050 may beformed as a sub-channel in layer 9518. In the illustrated embodiment,the vacuum way 9050 has an arch shape that is designed to hold channel9053 open and connect the channel 9053 to the piercings 9586 in thepierced layer 9585 and to the wells 9582, although other shapes arepossible. Without the formed vacuum way 9050, with certain films orother materials, channel 9053 may tend to “kiss” shut when a vacuum inapplied to the channel and prevent evacuation of the array. In oneembodiment, the vacuum channel 9050 may be formed in layer 9518 with aheat forming fixture (e.g., an appropriately shaped hot ‘debossing’wire). In other embodiments, the vacuum channel 9050 may be formed bylaser etching, xurography, or the like.

Pierced layer 9585 and second layer 9587 are plastic films that havebeen sealed to high density array 9581, illustratively by heat sealing(e.g., a heat-activated seal), although it is understood that othermethods of sealing may be employed. It is also understood that thematerial used for high density array 9581 and the material used forpierced layer 9585 and second layer 9587 should be compatible with eachother, with the sealing method, and with the chemistry being used. Whenused for PCR, examples of compatible plastics that can used for highdensity array 9581 and can be heat-sealed are PE, PP, Monprene®, andother block copolymer elastomers. If fluorescent dyes are used in thedetection chemistry, it may be desirable for high density array 9581 tobe formed from black or other relatively fluorescently opaque materials,to minimize signal bleed from one well 9582 to its neighboring wells andfor at least one of layers 9585 and 9587 to be relatively fluorescentlytransparent. For pierced layer 9585 and second layer 9587, laminates ofa strong engineering plastic such as Mylar® or PET with heat-sealableplastic layers such as PE, PP and Dupont Surlyn® may be used. Foradhesive-based systems, rigid engineering plastics such as PET orpolycarbonate may be used to form high density array 9581 and films ofPCR-compatible plastics are then used as pierced layer 9585 and secondlayer 9587. In one illustrative embodiment, high density array 9581 isformed of black PE, a composite polyethylene/PET laminate (or Xerox® PN104702 hot laminating pouch material) is used for pierced layer 9585 andsecond layer 9587 which are heat sealed to high density array 9581, andcomposite polypropylene/PET is used for first and second layers 9518,9519 of pouch 9510.

It is understood that piercings 9586 align with wells 9582. It is alsounderstood that piercings 9586 are small enough that, absent some force,fluid does not readily flow through piercings 9586. Illustrativepiercings may be 0.001-0.1 mm, more illustratively 0.005-0.02 mm, andmore illustratively about 0.01 mm. In an illustrative embodiment, avacuum is applied to second-stage amplification zone 9580 in situ bypulling a vacuum on wells 9582 through vacuum channel 9050 throughvacuum port 9051 and then sealing (e.g., heat sealing) the vacuumchannel 9050. Subsequently, when fluid is provided, the vacuum drawsfluid through piercings 9586 into each well 9582. Once the wells 9582are filled and pressure is equalized, a force is no longer present toforce fluid into or out of the wells 9582. Illustratively, wells may besealed after filling by applying a heat seal substantially perpendicularto the fill channel 9052. In another embodiment, a bladder adjacentsecond-stage amplification zone 9580 (not shown, but similar in positionto bladders 880/882) may then be activated to press first layer 9518against high density array 9581 and seal fluid into the wells 9582.While first layer 9518 of pouch 9510 is used to seal the wells 9582, itis understood that an optional sealing layer may be provided betweenpierced layer 9585 and first layer 9510.

In one illustrative example, second-stage amplification zone 9580 may beprepared as follows. High density array 9581 may be prepared by firstpunching, molding, or otherwise forming an array of wells 9582 in aplastic sheet (illustratively 0.1 to 1 mm thick). The wells may form anyregular or irregular array that is desired, and may have a volumeillustratively of 0.05 μl to 20 μl, and more illustratively of 0.1 μl to4 μl. A backing layer (e.g., 9587) is then laminated to a first surface9581 a of high density array 9581, illustratively by heat or adhesive.As shown in FIG. 9B, second layer 9587 is applied to first surface 9581a. Reagents 9589, illustratively elements of the chemistry of the arraythat are unique, such as PCR primer pairs, are then spotted into thewells either manually by pipetting, or automatically (illustrativelyusing x/y positionable spotters such as pin-spotters, dot-matrixprinters, small-volume automatic pipettes, or micro-fluidicmicro-contact spotters, for example spotters as taught in U.S. PatentApplication No. 2017-0209844, herein incorporated by references). Afterthe reagents 9589 have been dried in each well 9582, pierced layer 9585,which has been previously bonded to layer 9518, may be applied to thesecond surface 9581 b of array 9581. Array 9581 may be bonded to layer9519 of pouch 510 and sealed in place, illustratively by either heatsealing, using an adhesive, ultrasonically welding, mechanical closure,or other means of enclosing array 9581 inside pouch 510 within blister580. It is understood that second-stage reaction zone 9580 is fluidlyconnected to a downstream fluid blister (e.g., a first stage reactionzone) via channel 9565, and that liquid can flow from channel 9565 intosecond-stage reaction zone 9580 and over piercings 9586 through channel9565, which is in fluid communication with fluid flow channel 9053 andvacuum way 9050.

Second-stage reaction zone 9580 may be used in a manner similar tosecond-stage array 11000 of FIG. 7A or array assembly 8000 a of FIG. 7A.Because a vacuum may be applied to array 9581, when liquid is moved fromblister a downstream fluid reservoir to second-stage amplification zone9580, the liquid sample is drawn through fluid flow channel 9053 andpiercings 9586 and into wells 9582.

Because the array assembly is provided with a vacuum port in fluidcommunication with the plurality of wells, a vacuum can be applied(locally) to the array assembly (rather than (globally, duringmanufacture or assembly of an analytical device, such as a pouch inwhich the array assembly is disposed). Accordingly, an evacuationchamber is not required to apply a vacuum during manufacture, becausethe product need not be assembled under vacuum conditions. Instead, apiston pump (e.g., a syringe or syringe-type vacuum device) or anothervacuum device can be used to apply a vacuum on-demand, at thepoint-of-use, and even during performance of other steps of ananalytical method, rather than during assembly of an analytical device,such as a pouch in which the array assembly is disposed. Accordingly, anevacuation chamber is not required to apply a vacuum so that the productcan be assembled under vacuum conditions. Instead, a piston pump orother vacuum device can be used to apply the vacuum.

In one illustrative manufacturing process, the volume of an evacuationchamber used during manufacture of the analytical device comprising thearray assembly that is packages under vacuum would be many, many timeslarger than the volume of the wells and channel system of the arrayassembly. Evacuating larger volumes requires a larger pump and/or takeslonger than a smaller volume. Larger pumps, such as a diaphragm vacuumor rotary vane pump, are generally worse at pulling a (hard) vacuum in alarger volume than is a piston pump at pulling a (hard) vacuum in asmaller volume. Moreover, larger pumps capable of pulling the level ofvacuum pulled by a piston pump can be much more expensive to acquire,operate, and/or maintain than a piston pump. In addition, a singlestroke of a piston pump can pull a (level of) vacuum that would takemuch longer for a larger, diaphragm or rotary vane pump. Accordingly, ina given amount of time, a piston pump can pull a more complete (e.g.closer to a total) vacuum than can a larger, diaphragm or rotary vanepump.

As described above, the vacuum can also be applied on-demand, such as atthe point-of-use. Accordingly, the vacuum need not be held or maintainedby the array assembly for an extended period of time. Instead, unlikeexisting systems where the analytical device is packaged under vacuum,such that the analytical device must hold or maintain the vacuum duringstorage, shipping, transfer to the analytical device, and preliminarysteps in an analytical process, the on-demand vacuum can be applied tothe wells moments before and/or maintained right up until releasing orusing the vacuum to draw fluid into the wells (e.g., through the channelsystem) by opening the fluid seal. For instance, in a preferredembodiment, the vacuum need only be maintained by the by the arrayassembly for a few seconds (i.e., long enough to create a vacuum sealand open the fluid seal), before the liquid sample is drawn into thewells. In this way, as much as (nearly) the full level of the drawnvacuum can be used to draw a full, consistent amount of fluid into eachof the wells upon opening the fluid seal to release the vacuum.

In addition, because the vacuum is not drawn through the liquid samplevia the fluid opening, the level or strength of the vacuum is notlimited to the partial pressure of the fluid sample. The negativepressure level of this stronger vacuum (e.g., illustratively between 2mBar and 140 mBar) can thoroughly evacuate air from the array assemblyso that residual air is minimized in the array assembly, which minimizesair bubbles that may be trapped by the drawn fluid in the channels andwells or the array assembly. Moreover, the array assembly can beconfigured (e.g., sized and structured) so that an evacuation chamber isnot required to apply the vacuum. Instead, the combined volume of thewells and channel system can be sufficiently small to allow all orsubstantially all of the air to be evacuated with a single stroke of apiston pump. It will be appreciated, however, that other types of vacuumdevices (e.g., rotary vane or diaphragm pumps) can be used in someembodiments.

FIG. 11A schematically illustrates a system 1100 that includes aninstrument 1101 that can draw a vacuum in a reaction container in situwhile performing one or more steps of an analytical method. An exemplaryreaction container that is configured for in situ application of avacuum to one or more parts (e.g., blisters, the second-stage array,reagent wells or blisters, etc.) of the reaction container is shown at1102. While reaction container 1102 has a specific form and layout, onewill appreciate that this is merely exemplary and that other reactioncontainers—e.g., pouch 8000 a—may be adapted for in situ application ofa vacuum.

Reaction container 1102 includes a sample blister 1103 that may be usedfor introducing a sample into the reaction container. In one embodiment,the sample may be introduced with a swab 1104. However, swab 1104 ismerely illustrative. In other embodiments, a liquid sample may beintroduced with, for example, a transfer pipet, or a dry sample (e.g., asoiled tissue) may be placed into the sample blister 1103. The reactioncontainer 1102 further includes a lysis zone 1105 where cells andviruses in the sample can be lysed to free their nucleic acids foranalysis and a reaction zone 1106 where various method steps can beperformed such as, but not limited to, nucleic acid recovery andpurification, first-stage multiplex PCR, and preparation of first-stageproduct for second-stage PCR. In one embodiment, the lysis zone 1105 andthe reaction zone 1106 can be combined into one zone for lysis,purification of nucleic acids, first-stage PCR, etc. In anotherembodiment, the lysis zone 1105 and the reaction zone 1106 can bedivided between a number of separate blisters dedicated to separatefunctions. Pouch 510 is an example of such a reaction container with anumber of separate blisters dedicated to different functions.

Illustrative reaction container 1102 further includes a number ofreagent blisters 1107 a-1107 d that can be used to provide reagents forassays run in the reaction container. The reagent blisters can containdry reagents, liquid reagents, or a combination of both. While fourreagent blisters are shown, one will appreciate that more or fewer maybe included, depending on the assay to be performed in the reactioncontainer. Likewise, the reagent blisters may be connected to thereaction blisters in a number of different configurations, depending onthe assay to be performed in the reaction container. Reaction container1102 further includes a number of channels 1109 a-1109 g used to connectthe zones of the reaction container. In one embodiment, the channels1109 a-1109 g may be openably sealed, as explained in detail elsewhereherein. Reaction container 1102 further includes an array 1181 thatincludes a number of wells 1182 that can be configured for individualassay reactions.

While instrument 1101 is shown only schematically, persons of ordinaryskill will appreciate that instrument 1101 may be configured forperforming a number of manipulations, heating steps, cooling steps, etc.on reaction container 1102 in the process of performing an analyticalmethod. In one embodiment, instrument 1101 may include one or moreheaters configured for controlling the temperature of one or moreportions of reaction container 1102. For instance, such heaters may beconfigured for thermocycling one or more portions of reaction container1102 for performing one or more PCR reactions. Similarly, such heatersmay be configured for one or more isothermal processes in one or moreportions of reaction container 1102. In one embodiment, instrument 1101may include a lysis apparatus (e.g., a bead beater like element 804 ofFIG. 2, a paddle bead beater, a sonicator, etc.) for performing celllysis in one or more portions of reaction container 1102. In oneembodiment, instrument 1101 may include a magnet for magnetic beadcapture. In one embodiment, instrument 1101 may include actuators formoving fluids within one or more portions of the reaction container1102. In one embodiment, instrument 1101 may include one or more seals(e.g., retractable seals and/or heat seals) for controlling the movementof fluids within one or more portions of the reaction container 1102. Inone embodiment, instrument 1101 may light source(s) and image capturesystems for optical (e.g., fluorescent) excitation and data collectionfrom one or more portions of the reaction container 1102.

In addition, instrument 1101 may include a computer (either an internalor external computer, or both), an integrated display, a heat sealapparatus to apply heat seals to one or more portions of the reactioncontainer 1102, and/or compression members (e.g., inflatable bladders)for compressing one or more portions of the reaction container 1102against components of instrument 1101. For example, compression membersmay be used to improve thermal contact between one or more portions ofthe reaction container 1102 and one or more heaters. In one embodiment,instrument 1101 is configured for performing at least one PCR reactionin the reaction container 1102. Other components that may be included ininstrument 1102 are shown in FIGS. 2-4 and are discussed in theaccompanying text. However, one will appreciate that the form andarrangement of the components shown in FIGS. 2-4 may be different ininstrument 1102, but the functions may be the same or similar.

In addition to the foregoing, the instrument 1101 and the reactioncontainer 1102 may be configured for drawing a vacuum in one or moreportions of reaction container 1102 either prior to or while performingan analytical method. As discussed in detail elsewhere herein, havingone or more portions of reaction container 1102 under partial vacuum canfacilitate filling one or more portions with a fluid. For example,filling the array wells 1182 may be greatly facilitated by having thearray 1181 under partial vacuum.

In one embodiment, the reaction container 1102 includes a channel systemsimilar to those illustrated, for example in FIGS. 7A, 8A, 8C, and 9Afor applying a vacuum to one or more portions of the reaction container.In addition, instrument 1101 includes a system for drawing a vacuum inone or more portions of the reaction container 1102 while performing ananalytical method. The vacuum system includes a vacuum line 1110 and avacuum source 1112. Vacuum source 1112 may be essentially any vacuumsource or vacuum generating device known in the art. Examples include,but are not limited to, piston pumps (e.g., a syringe pump), positivedisplacement pumps, momentum transfer pumps (also called molecularpumps), and entrapment pumps (also called molecular traps). In oneembodiment, vacuum source 1112 may include an evacuated chamber that isconnected to a vacuum pump.

In such a system, the partial vacuum in the evacuated chamber may beused to draw a vacuum on one or more portions of the reaction container1102 and, in turn, a vacuum pump may be used to draw or maintain avacuum in the evacuated chamber. In one embodiment, vacuum source 1112and vacuum line 1110 may draw a vacuum on array 1181 at vacuum port1108. In one embodiment, vacuum source 1112 may be fluidly coupled to atleast one additional vacuum line for drawing a vacuum on at least oneadditional portion of the reaction container 1102. For example, FIG. 11Aillustrates an extension 1118 on vacuum line 1110 that may be used fordrawing a vacuum on at least one reagent blister via port 1116.

In one embodiment, the instrument 1101 may be used to apply a vacuum toone or more portions of the reaction container 1102 while performing ananalytical method, or after the instrument has performed at least onestep of an analytical method. For instance, the instrument 1101 may drawa vacuum in the reaction container 1102 while or after inserting thereaction container into the instrument, while or after hydrating asample, while or after performing one or more method steps or reactionsin a first reaction zone (e.g., the lysis zone 1105), while or afterperforming one or more method steps or reactions in a second reactionzone (e.g., reaction zone 1106), or in preparation for filling one ormore portions of the reaction container 1102 with fluid. For instance,instrument 1101 may draw a vacuum in the array 1181 or in reagentblister 1107 d (or another reagent blister) in preparation for fillingthem with fluid.

In one example, the one or more method steps or reactions in a firstreaction zone may include, but are not limited to, sample lysis (e.g.,by bead beating), isolation of lysis particles from the lysate, movingthe lysate to another chamber in the reaction container, mixing silicamagnetic beads with the lysate, or capturing the magnetic beads andmoving them to another chamber in the reaction container. In oneexample, the one or more method steps or reactions in a second reactionzone may include, but are not limited to, mixing silica magnetic beadswith a lysate from the first reaction zone, moving the spent lysate(i.e., residual lysate after nucleic acid capture) to another chamber inthe reaction container (e.g., to a waste chamber), performing at leastone wash of the magnetic beads and moving the wash liquid to anotherchamber in the reaction container, elution of nucleic acids from thesilica magnetic beads, performing a first PCR reaction (e.g., asingleplex or a multiplex PCR), or diluting a product of the first PCRreaction in preparation for performing a second PCR reaction in thewells of the array.

FIGS. 11B and 11C illustrate systems for drawing of a vacuum on an arrayand a reagent blister in greater detail. FIG. 11B shows a vacuummanifold 1128 and 1130 for an array 1181. Details of an embodiment of avacuum manifold and how it relates to the array, the array wells, etc.was shown in FIGS. 8A-8C and discussed in the accompanying text. In theillustrated embodiment, the vacuum line 1110 and vacuum source 1112connect to the vacuum port 1108 via vacuum hub 1111. Vacuum hub 1111 maybe a resilient member like a suction cup, a nipple, or the like known inthe art. In one embodiment, vacuum hub 1111 may include a sharp member(e.g., a needle or the like) positioned to pierce a layer of film overvacuum port 1108. In another embodiment, vacuum port 1108 may include aflapper valve or other valve so that vacuum port may normally be sealedfrom the outside but the valve can open to allow air to escape when avacuum is drawn on vacuum port 1108. After a vacuum is drawn on thearray 1181, a seal (e.g., a heat seal) may be applied in the region of1164 to seal channels 1128 and 1130 from outside air and to preserve thevacuum in the array after vacuum hub 1111 is removed.

FIG. 11C shows an illustrative blister 1107 d that may be configured forhaving a vacuum drawn in situ. And while one reagent blister 1107 d isshown, one will appreciate that multiple reagent blisters may be linkedto one vacuum system, or a reaction container like 1102 may include twoor more reagent blisters that may be configured for having an in situapplied vacuum. In the illustrated embodiment, the vacuum line 1118 andvacuum source 1112 connect to reagent blister 1107 d via vacuum port1116 and reaction container vacuum line 1114 via vacuum hub 1117. Aswith vacuum hub 1111, vacuum hub 1117 may be a resilient member like asuction cup, a nipple, or the like known in the art. In one embodiment,vacuum hub 1117 may include a sharp member (e.g., a needle or the like)positioned to pierce a layer of film over vacuum port 1116. In anotherembodiment, vacuum port 1116 may include a valve so that vacuum port maynormally be sealed from the outside but the valve can open to allow airto escape when a vacuum is drawn on vacuum port 1108. Reaction containervacuum line 1114 may be formed as described elsewhere herein to keep theline open when a vacuum is applied. For instance, reaction containervacuum line 1114 may include an embossed arch formed in the film used tofabricate reaction container 1102, the reaction container vacuum line1114 may be formed in a card material. As discussed elsewhere herein, anopenable seal 1121 in channel 1109 e may prevent the vacuum applied toblister 1107 d from being applied to other regions of the reactioncontainer 1102. After a vacuum is drawn on reagent blister 1107 d, aseal (e.g., a heat seal or a cap) may be applied in the region of 1122to preserve the vacuum in reagent blister 1107 d after vacuum hub 1117is removed.

In one embodiment, reagent blister 1107 d may include a dried chemistry1120 that may need to be rehydrated for use in an assay. It may beadvantageous to apply a partial vacuum to reagent blister 1107 d inorder to, for example, allow rehydration fluid to flow into reagentblister 1107 d without air blocking fluid flow and so that bubbles arenot subsequently introduced into reaction chambers (e.g., reactionchamber 1106). The dried chemistry 1120 may be air dried or freeze driedand may include reaction components (e.g., enzymes), buffers,stabilizing agents, and the like. The dried chemistry 1120 may be in theform of a powder, a dried reagent pill, a reagent spotted onto a filterpaper, or other forms known in the art.

It is understood that in various embodiments of arrays described herein,reaction components may be spotted in the wells. For example, primersmay be spotted in each well for priming nucleic acid amplificationreactions. Each well may have a different pair of primers, various wellsmay include replicates of primers found in other wells, or anycombination thereof. Additional materials may be spotted, including oneor more of nucleotide tri-phosphates (NTPs), polymerase, magnesium, orother components. When such components are spotted and dried duringmanufacturing, the components often dry around the edges of the well,leaving a relatively small surface area for rehydration. With fast PCRembodiments, see, e.g. PCT/US2017/18748, herein incorporated byreference, the time needed for such rehydration may extend through anumber of PCR cycles, which may require additional cycling time.

Turning now to FIGS. 10A-F, one way to decrease the time spent forrehydration and dissolution during second-stage PCR thermal cycling isto flood the array with a hydration fluid prior to the time in thereaction process when the array will be used, illustratively duringsample preparation or during first-stage PCR. FIGS. 10A-10E illustratean example of a method for rehydrating the contents and initiating areaction in an illustrative well of a second-stage array. FIG. 10Fillustrates an alternative method of initiating a reaction in the methodillustrated in FIGS. 10A-10E. While FIGS. 10A-F show one well 1082 of anarray 1080, it is understood that a plurality of wells may be present,illustratively in arrangements such as in array 580, array 5081, andarray 8000, as well as other array configurations. Illustrative well1082 is formed in array layer 1081, and is bounded by pierced layer 1085and second layer 1087. It is understood that layer 1018 may be an outerlayer of a pouch, similar to layer 518, or layer 1018 may be an extraflexible layer provided for rehydration methods. Array 1080 may be usedin various embodiments described herein or in other sample processingsystems.

Array 1080 may be spotted with various components 1040 for sampleprocessing, illustratively components for PCR. Illustrative ways ofspotting array 1080 are described in U.S. Pat. Pub. No. 2015/0283531,herein incorporated by reference, but it is understood that other waysof spotting array 1080 are within the scope of this disclosure.Subsequent to spotting, array 1080 may be dried. As illustrated in FIG.10A, the components 1040 often dry into the corners of well 1082.Optionally, array 1080 may be evacuated at point of use, as shown by thearrow in FIG. 10A and as described in detail elsewhere herein. Likewise,array 1080 may be evacuated at the time of manufacture and then storedunder vacuum until time of use.

In FIG. 10B, hydration fluid 1070, illustratively water or buffer, isintroduced to well 1082 through opening 1086. It is understood thatopening 1086 may be a piercing, such as piercing 586 described elsewhereherein, where opening 1086 is small enough that, absent some force,fluid does not readily flow through opening 1082. In some embodiments,however, it may be desirable for opening 1086 to be larger, or it may bedesirable to use other barrier layers. After filling with hydrationfluid 1070, excess hydration fluid may be removed from the array,illustratively by rolling the fluid toward a waste receptacle (notshown) using roller 1050, as shown by FIG. 10C, or by placing pressureon the array, for example by a bladder located in an instrument adjacentarray 1080. Since the materials in each array well are in need ofrehydration, if excess hydration fluid 1070 is removed immediately, theamount of cross-contamination between wells will be minimized, as theexcess hydration fluid is removed as the reaction components 1040 beginto rehydrate into the hydration fluid 1070. Optionally, pressure may beplaced on layer 1087 as excess hydration fluid 1070 is moved to thewaste receptacle, which pressure reduces the volume of well 1082,thereby causing well 1082 to be under-filled.

In FIG. 10D, subsequent to first-stage PCR, the first-stage reactionmixture 1045 may be moved into array 1080, illustratively by pressure.Because each of the wells are flexible and are already will be filled,but illustratively not over-filled due to the removal of excesshydration fluid, each well can accept a small amount of reactionmaterial such as first-stage PCR reaction mixture 1045. Because only asmall amount of first-stage PCR reaction mixture 1045 is introduced toeach well, a dilution step of the first-stage PCR reaction mixture priorto introduction of the first-stage PCR reaction mixture may be omitted.If well 1082 is under-filled as discussed above, the amount first-stagePCR reaction mixture 1045 that may be introduced into each well may besomewhat greater, and, accordingly, the amount of dilution will besomewhat reduced. The amount of pressure placed on layer 1087 duringremoval of excess hydration fluid 1070 may be adjusted to provide anappropriate amount of dilution.

As shown in FIG. 10E, roller 1050 or other pressure may be used again toremove excess first-stage PCR reaction mixture 1045. In thisillustrative embodiment, only a small amount of first-stage PCR reactionmixture 1045 is pushed through opening 1086. FIG. 10F shows analternative embodiment, where instead of forcing excess first-stagereaction mixture 1045 out of array 1080, layer 1018 is sealed to piercedlayer 1085, illustratively by pressure or heat sealing at arrows 1090.In this embodiment, a bolus of first-stage PCR reaction mixture 1045becomes sealed outside opening 1086, and first-stage PCR reactionmixture 1045 and components 1040 may continue to mix during subsequentthermal cycling. This embodiment may result in less dilution than theembodiment shown in FIG. 10E.

Because this pre-rehydration of the array 1080 may include relevantreactants, it may be desirable to omit one of the reactants from thearray until the first-stage reaction mixture is provided to the array sothat the reaction mixture is incomplete and the reaction in the arraycannot begin until the first-stage reaction mixture is provided. Forexample, if the second-stage array 1080 is used for PCR, magnesium maybe omitted from both the dried array components and the hydration fluid,thus preventing primer dimers from forming during rehydration. In such amethod, high concentration of magnesium may be added to the first-stagereaction mixture prior to providing the first-stage reaction mixture tothe array, and dilution of this mixture when the small amount of thefirst-stage reaction mixture is introduced into each well provides theappropriate concentration of magnesium to the array to allow PCR toproceed. It is understood that magnesium is an illustrative component,and that other components may be used to control start of amplification.Also, it is understood that a complete mixture may be provided to eachwell, and that the start of the reaction may be controlled bycontrolling temperature of the reactions, illustratively by coolingusing a heater such as heater 888.

Accordingly, embodiments of the present disclosure provide numerousadvantages over existing systems.

CONCLUSION

While the foregoing detailed description makes reference to specificexemplary embodiments, the present disclosure may be embodied in otherspecific forms without departing from its spirit or essentialcharacteristics. Accordingly, the described embodiments are to beconsidered in all respects only as illustrative and not restrictive. Forinstance, various substitutions, alterations, and/or modifications ofthe inventive features described and/or illustrated herein, andadditional applications of the principles described and/or illustratedherein, which would occur to one skilled in the relevant art and havingpossession of this disclosure, can be made to the described and/orillustrated embodiments without departing from the spirit and scope ofthe invention as defined by the appended claims.

The limitations recited in the claims are to be interpreted broadlybased on the language employed in the claims and not limited to specificexamples described in the foregoing detailed description, which examplesare to be construed as non-exclusive and non-exhaustive. All changeswhich come within the meaning and range of equivalency of the claims areto be embraced within their scope.

It will also be appreciated that various features of certain embodimentscan be compatible with, combined with, included in, and/or incorporatedinto other embodiments of the present disclosure. For instance, systems,methods, and/or products according to certain embodiments of the presentdisclosure may include, incorporate, or otherwise comprise featuresdescribed in other embodiments disclosed and/or described herein. Thus,disclosure of certain features relative to a specific embodiment of thepresent disclosure should not be construed as limiting application orinclusion of said features to the specific embodiment. In addition,unless a feature is described as being requiring in a particularembodiment, features described in the various embodiments can beoptional and may not be included in other embodiments of the presentdisclosure. Moreover, unless a feature is described as requiring anotherfeature in combination therewith, any feature herein may be combinedwith any other feature of a same or different embodiment disclosedherein.

1.-13. (canceled)
 14. A method of using an array assembly on a sample, comprising (a) providing a sample container comprising a reaction zone in fluid communication with the array assembly, the array assembly comprising a plurality of wells configured in an array, an access opening between the reaction zone and the array, a vacuum port, and a plurality of channels such that each well in the array is fluidly connected to the access opening and the vacuum port; (b) performing an analytical method on the sample in the reaction zone to produce a reaction mixture; (c) opening the vacuum port and drawing a vacuum on the array assembly such that air is evacuated from the plurality of wells and the plurality of channels; (d) sealing the vacuum port such that the plurality of wells are maintained under the vacuum, thereby forming an evacuated array; and (e) opening the access opening such that the reaction mixture is drawn into the plurality of wells via the plurality of channels.
 15. The method of claim 14, further comprising mixing the reaction mixture with one or more reagents disposed in each of the plurality of wells to form a second mixture, and performing a second analytical method on the second reaction mixture.
 16. The method of claim 15, wherein the second mixture is a PCR mixture.
 17. The method of claim 16, further comprising thermocycling the second reaction mixture.
 18. The method of claim 17, further comprising detecting an amplification product in at least one of the plurality of wells.
 19. The method of claim 15, wherein the sample comprises a microorganism and the one or more reagents comprises an antibiotic.
 20. The method of claim 19, wherein the antibiotic is present in a first of the plurality of wells at a first concentration and is present in a second of the plurality of wells at a second concentration, wherein the second concentration is lower than the first concentration.
 21. The method of claim 14, wherein step (c) provides a vacuum in the plurality of wells of between about 2 millibar and about 150 millibar.
 22. The method of claim 14, wherein steps (c) and (d) are performed before step (b) is completed. 23.-51. (canceled)
 52. A method of drawing a vacuum on a reaction container in situ while performing an analytical method, the method comprising: providing a reaction container that includes a sample introduction zone, at least a first reaction zone in fluid communication with the sample introduction zone, and a second reaction zone in fluid communication with the first reaction zone, wherein the second reaction zone comprises a plurality of wells, a vacuum channel in fluid communication with the plurality of wells and a vacuum port, an array fill channel extending between and/or in fluid communication with the plurality of wells and the first reaction zone, and an openable seal disposed between the first reaction zone and the second reaction zone; performing at least one step of the analytical method with the reaction container; drawing a partial vacuum on the plurality of wells such that the plurality of wells, the vacuum channel, and at least a portion of the array fill channel are under reduced pressure relative to atmospheric pressure; sealing a portion of the vacuum channel such that the plurality of wells and at least the portion of the array fill channel are maintained under the vacuum, thereby forming an evacuated array; and applying a fluid to the evacuated array by opening the openable seal disposed between the first reaction zone and the second reaction zone such that the fluid is drawn into the plurality of wells via the array fill channel.
 53. The method of claim 52, wherein the at least one step of the analytical method includes removing the reaction container from an ambient pressure package, introducing a sample into the sample introduction zone, inserting the reaction container into an instrument configured for performing the analytical method and configured for drawing a partial vacuum to form the evacuated array, performing one or more reactions in the first reaction zone, or preparing for applying the fluid to the evacuated array.
 54. The method of claim 53, wherein performing one or more reactions in the first reaction zone comprises one or more of performing a sample lysis to generate a lysate, isolating lysis particles from the lysate, mixing silica magnetic beads with the lysate, moving a residual lysate after nucleic acid capture with the silica magnetic beads to a waste chamber, performing at least one wash of the magnetic beads and moving the wash liquid to the waste chamber, eluting nucleic acids from the silica magnetic beads, performing a first singleplex or multiplex PCR reaction, or diluting a product of the first PCR reaction in preparation for performing a second PCR reaction in the plurality of wells of the second reaction zone.
 55. The method of claim 52, wherein the reaction container further includes one or more reagent blisters fluidically connected to one or more of the sample introduction zone, the first reaction zone, or the second reaction zone.
 56. The method of claim 55, wherein at least one of the one or more reagent blisters includes a dried reagent, and the method further comprising drawing a partial vacuum on the one or more reagent blisters including the dried reagent.
 57. The method of claim 56, further comprising mixing a fluid with the dried reagent to form a first mixture.
 58. The method of claim 56, wherein the one or more reagent blisters comprise reagents for sample preparation, nucleic acid recovery, a first-stage PCR, and a second-stage PCR.
 59. A system, comprising: a reaction container that includes a sample introduction zone, at least a first reaction zone in fluid communication with the sample introduction zone, and a second reaction zone in fluid communication with the first reaction zone, wherein the second reaction zone comprises a plurality of wells, a vacuum channel extending between and/or in fluid communication with the plurality of wells and a vacuum port, an array fill channel extending between and/or in fluid communication with the plurality of wells and the first reaction zone, and an openable seal disposed between the first reaction zone and the second reaction zone; and an instrument configured to perform an analytical method using the reaction container, wherein the instrument includes a vacuum system to draw a partial vacuum in one or more portions of the reaction container while performing one or more steps of the analytical method.
 60. The system of claim 59, wherein the one or more portions of the reaction container configured to have a partial vacuum drawn thereon are substantially dry such that the partial vacuum is not being drawn against a partial pressure of water.
 61. The system of claim 60, wherein the partial vacuum in the one or more portions of the reaction container configured to have a partial vacuum drawn thereon is in a range between about 2 millibar and about 150 millibar.
 62. The system of claim 59, wherein the one or more portions of the reaction container configured to have a partial vacuum drawn thereon include the plurality of wells, the vacuum channel, and at least a portion of the array fill channel.
 63. The system of claim 59, wherein the instrument includes a seal device to apply a seal to preserve the partial vacuum drawn in the one or more portions of the reaction container.
 64. The system of claim 59, wherein the reaction container further includes one or more reagent blisters fluidically connected to one or more of the sample introduction zone, the first reaction zone, or the second reaction zone.
 65. The system of claim 64, wherein at least one of the one or more reagent blisters includes a dried reagent, and wherein the one or more portions of the reaction container configured to have a partial vacuum drawn thereon include the one or more reagent blisters that include the dried reagent.
 66. The system of claim 59, wherein the instrument is a PCR instrument that includes at least one heater positioned and arranged for thermocycling at least one portion of the reaction container.
 67. The system of claim 59, wherein the instrument includes at least one heater positioned and arranged for controlling the temperature in at least one portion of the reaction container for performing an isothermal reaction.
 68. The system of claim 59, wherein the instrument includes at least one heater positioned and arranged for controlling the temperature in at least one portion of the reaction container, one or more actuators for moving fluid in the reaction container, and one or more seals for controlling the movement of fluids within one or more portions of the reaction container. 